Protein chips, preparation and use thereof

ABSTRACT

The present invention relates to the field of medicine, in particular of research and diagnosis. It relates more particularly to a novel tool for detecting antibodies in a biological sample originating from a mammal. This tool, which is in the form of a protein chip, can be used in screening for new targets of interest involved in the occurrence of an autoimmune disease, in particular of a disease affecting the nervous system of a mammal, and also in the diagnosis or the monitoring of the progression of such an autoimmune disease. The invention also relates to a method for producing such a tool and also to kits comprising it and enabling its use.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of International Patent Application No. PCT/EP2013/052009, filed Feb. 1, 2013.

The Sequence Listing for this application is labeled “Seq-List.txt” which was created on Jul. 25, 2014 and is 2,557 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.

The present invention relates to the field of medicine, in particular of research and diagnosis. It relates more particularly to a novel tool for detecting autoantibodies in a biological sample originating from a mammal. This tool, which is in the form of a protein chip, can typically be used in screening for new targets of interest involved in the occurrence of an autoimmune disease, in particular of a disease affecting the nervous system of a mammal, and in the diagnosis or the monitoring of the progression of such an autoimmune disease. The invention also relates to a method for producing such a tool and also to kits comprising it and enabling its use.

PRIOR ART

The prior art provides several types of tests for detecting the presence of a target molecule within a sample. Among these are radioimmunoassays (RIAs); immunofluorescence assays (IFAs); luminescence immunoassays (LIAs); EIA assays (enzyme immunoassays), in particular ELISA assays (enzyme-linked immunosorbent assays), i.e., enzymatic immunoassays on a solid support.

MA (radioimmunoassay) makes it possible to measure antigen concentrations in a sample using antibodies labelled with a radioelement. The assay of the radioelement measures a number of disintegrations per second. This technique is the reverse technique of the RBA (“radiobinding assay”) which makes it possible to quantify an antibody by means of the corresponding antigens. These techniques are simple to carry out and inexpensive, but require the use of radioactive substances, often iodine isotopes bonded to tyrosine residues. Radioimmunoprecipitation assays (RIPAs) enable the precipitation of an antigen of interest present in a sample using a labelled specific antibody. This method can be used to isolate and concentrate a particular protein within a sample comprising thousands of other proteins. The technique requires the antibody to be coupled to a solid substrate.

Immunofluorescence assays (IFAs) use antibodies (or immunoglobulins) and also fluorochromes (chemical substances capable of emitting fluorescent light after excitation). The fluorochromes most commonly used are phycoerythrin (PE), fluorescein isothiocyanate (FITC), the Alexa Fluor range, and green fluorescent protein (GFP). These assays have the advantage of giving results very rapidly, or even immediately, dispensing with the long exposure times required for the technique with radioactivity. Fluorescence has the drawback of not being permanent, the intensity of the fluorescence decreasing over time until it becomes undetectable. In the context of luminescence immunoassays (LIAs), the enzymatic activity is measured by luminometry.

The ELISA (enzyme-linked immunosorbent assay) falls within the more general context of EIAs (enzyme immunoassays) in which the assaying of a reaction between antigen and antibody is coupled to a reaction catalyzed by an enzyme which releases a coloured component that is monitored by spectroscopy. The ELISA is a biochemical technique, mainly used in immunology in order to detect the presence of an antibody or an antigen in a sample (e.g., Gotti et al. Muscle & Nerve, 20: 800-808, 1997; Franciotta et al., Clin Chem. 45: 400-5, 1999; Hewer et al., Clin Chim Acta. 364:159-66, 2006). The technique uses one or two antibodies. One of them is specific to the antigen, while the other reacts with the immune (antigen-antibody) complexes and is coupled to an enzyme. This secondary antibody, which is responsible for the name of the technique, can also cause the emission of a signal by a chromogenic or fluorogenic substrate.

The assays available and described above are not, however, sensitive enough to detect very low concentrations of target molecules, typically of antibodies, in particular of autoantibodies and even more particularly of autoantibodies directed against membrane proteins and/or membrane protein complexes.

Moreover, they must generally be used on small amounts of serum. A dilution thus often proves to be necessary in order to avoid an excessive background noise. ELISA assays, for example, can require an at least 50-fold dilution of the serum. Radioimmunoprecipitation assays (RIPAs) prefer the use of only a few microliters (approximately 5 microliters) of test serum in order to avoid the use of large volumes of antiserum generating unacceptable background noises (i.e., inappropriate values of the radioactivity of the negative controls) owing to excessively large pellets. Even though some specific ELISA assays may use larger serum volumes (e.g., 50 to 100 microliters), their use is, however, limited and their sensitivity is not increased. For example, a commercial ELISA kit from RSR Ltd for detecting anti-nicotinic acetylcholine receptor (AChR) antibodies associated with myasthenia gravis allows the use of a combination of three anti-AChR monoclonal antibodies in a sandwich-type assay using 100 microliters of serum. However, this technique does not increase the sensitivity of the assay compared with the standard RIA assay. In addition, according to Irani et al. (Autoimmunity, February 2008; 41(1): 55-65), the ELISA technique, just like the “Western blotting” and “phage display” techniques, allows the identification of autoantibodies directed against non-conformational epitopes, but not that of autoantibodies directed against functional epitopes. The “Western blotting” technique, like immunohistochemistry, in particular involves the denaturation of the proteins of interest (H. Prüss et al: “Anti-NMDA-receptor encephalitis”, Der Nervenartz, vol. 81, no. 4, 31 Jan. 2010, pp. 396-408).

A certain number of recent studies (Vincent et al., Autoimmune channelopathies and related neurological disorders. Neuron 2006 52:123-138, and Vernino et al., Autoimmune encephalopathies. Neurologist 2007 13(3):140-7) show the presence and the involvement of autoantibodies in pathological conditions affecting the nervous system. The notion that neurons can be the target of an autoimmune attack, in particular in the central nervous system, is quite recent. Graus et al. (J. Antibodies and neuronal autoimmune disorders of the CNS. J. Neurol. 2010 257(4):509-17) suggest the detection of autoantibodies for diagnosing paraneoplastic neurological syndromes. Irani et al. (Autoantibody-mediated disorders of the central nervous system, Autoimmunity 2008 41(1):55-65) describe the existence of autoantibodies directed against ion channels and surface receptors having pathogenic effects in myasthenia gravis and acquired neuromyotonia. The pathogenicity of these antibodies is supported by the fact that encephalitis and paraneoplastic neurological syndromes (PNSs) respond positively to immunomodulatory treatments and to plasmapheresis. Furthermore, this positive therapeutic response correlates with a decrease in the titre of these autoantibodies assayed in the cerebrospinal fluid.

From a practical point of view, the regular description of new autoantibodies and the spectrum of associated syndromes are such that neurologists still have trouble estimating which antibodies or which set of antibodies is of use for diagnosing a given syndrome. The importance and the potential of such markers for diagnosis and treatment are, however, in no doubt, and require the development of systematic methods for detecting and characterizing these antibodies in order to meet the need for very substantially improving correlations between clinical syndrome, immunological signature and therapeutic response. Among the pathological conditions affecting the nervous system (neurological disorders or disease) which do not, at the current time, benefit from an adequate method of diagnosis are, in particular, encephalopathies, in particular those associated with neuronal excitability disorders.

There is, in fact, at the current time no tool which is sufficiently sensitive and specific to be able to effectively detect the autoantibodies, often present in very small amounts, directed against membrane proteins or membrane protein complexes, in particular proteins or complexes comprising functional epitopes, described as being involved, or suspected of being involved, in the occurrence of these encephalopathies in humans. Thus, for example, WO 2011/142900, which describes a method for the diagnosis of a neurodegenerative disease comprising the detection of autoantibodies and also a chip fabricated using proteins, specifies, however, that these proteins are produced in insect cells, and not, contrary to the invention, in mammalian cells, before being purified. Such a production method inevitably has an impact on the post-translational modifications of the proteins thus produced and, therefore, the recognition of epitopes by possible autoantibodies. It is also impossible, using such a chip, to enable the coexpression of partner subunits (protein complex) at the surface of said chip. The other types of tests for identifying autoantibodies that are carried out at the current time generally require, for their part, the sacrifice of animals, typically rodents (rats, mice), since they are all carried out on cells, in particular on transfected cells, or on tissue sections (typically placed on glass slides).

SUMMARY OF THE INVENTION

The inventors presently describe, for the first time, a protein chip for detecting antibodies (in particular identifying new antibodies), preferably autoantibodies, even more preferably autoantibodies directed against membrane proteins and/or membrane protein complexes, in a biological sample. This chip comprises at least one solubilized protein of interest and is in the form of a support for detecting the complexes formed between antibodies, preferably autoantibodies, and epitopes specific for said protein and/or for said protein complex, it being possible for said specific epitopes to be sequential or conformational epitopes. In one preferred embodiment of the invention, said at least one solubilized protein of interest is a solubilized membrane protein of interest and the protein chip is a membrane protein chip. In one even more preferred embodiment, the solubilized membrane protein of interest is expressed in the cells of the nervous system of an animal, preferably of a human being, and is involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitation disorders.

A considerable advantage of the protein chip according to the invention is that, owing to the preservation by the protein of its native three-dimensional structure, it makes it possible to detect both sensitively and specifically any antibody present in a biological sample capable of binding to any one of the epitopes (sequential as well as conformational) formed by the protein(s) of interest expressed on said chip. The membrane protein of interest may be alone (membrane protein) or associated with one or more membrane or perimembrane auxiliary subunits (membrane protein complex). By virtue of its specificity, this chip allows, moreover, the use of large amounts of biological samples to be tested and does not impose, in the context of its use, any step of dilution of said biological sample. It is thus now possible to detect the presence of antibodies even when their concentration in the sample to be tested is very low.

A particular subject of the invention relates to a protein chip for the simple and effective identification of antibodies, preferably of autoantibodies, in a biological sample, said chip comprising at least one solubilized protein of interest, typically at least one solubilized membrane protein of interest, preferably chosen from an ion channel protein, a transporter protein and a membrane receptor protein which regulates the activity of an ion channel protein or of a transporter protein, said protein being optionally associated with one or more auxiliary subunits, and said chip being in the form of a support for detection of the complexes formed between antibodies, preferably autoantibodies, and (sequential and also conformational) epitopes specific for said protein, optionally associated with its auxiliary subunit(s). Preferably, said at least one protein or said at least one membrane protein complex is expressed in the nervous system of a human being and is preferably involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders.

Another subject of the invention relates to a protein chip comprising several different proteins of interest, typically several different membrane proteins of interest, typically at least 50 or at least 100 different proteins of interest, preferably between approximately 200 and approximately 400 different proteins of interest, for example approximately 250 or approximately 300 different proteins of interest, each protein of interest being optionally associated with one or more auxiliary subunits.

Moreover, the invention relates to a method for preparing a chip as described previously, and also the chip capable of being obtained by means of such a method, said method comprising the following steps of cloning the cDNA(s) of interest, of expressing the protein(s) (typically the membrane protein(s)) encoded by said cDNA(s) in cells in culture, of solubilizing the proteins expressed using a non-denaturing detergent which enables the solubilization of said proteins while at the same time preserving their native three-dimensional conformation and their ability to bind to a support, and of depositing said solubilized proteins on a support in order to obtain the chip of interest. Contrary to the invention, WO2004/108762 describes methods for preparing solubilized thyroid membrane which all require a fractionation step using salts in order to obtain a fraction enriched with proteins of interest, and also an ultracentrifugation step.

The invention also relates to the use of a chip according to the invention for screening for antibodies of interest, preferably for an antibody involved in the occurrence of an autoimmune disease, or for the diagnosis and/or the monitoring of the progression of an autoimmune disease.

Another subject of the invention relates, moreover, to a kit comprising a chip as described previously and, optionally, one or more reagents preferably chosen from a buffer for blocking the non-specific sites, a buffer allowing the association between antibodies and antigens (Ab/Ag), a washing buffer, a secondary antibody, one or more product(s) for detecting said secondary antibody or antibodies, and instructions for use.

DETAILED DESCRIPTION

Products

The invention relates to a protein chip for detecting antibodies, typically autoantibodies, preferably autoantibodies directed against membrane proteins and/or membrane protein complexes, in a biological sample, and also the method for obtaining same and the uses thereof. More specifically, the invention relates to a protein chip for detecting autoantibodies, in particular identifying new autoantibodies, in a biological sample. The inventors describe, for the first time, in the context of the present invention, a chip in the form of a support comprising at least one solubilized protein of interest (typically a solubilized membrane protein of interest), preferably a protein having retained its native three-dimensional structure (or conformation), and allowing the detection of the complexes formed between antibodies, typically autoantibodies, and epitopes (sequential or conformational), typically conformational epitopes, specific for said protein of interest.

The present invention allows, for the first time, the detection, which is at the same time sensitive and specific, of the complexes formed between autoantibodies and epitopes (sequential and conformational) of the membrane protein of interest (optionally associated with its auxiliary subunit(s)), preferably of a protein or of a protein complex involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders. In addition, this method makes it optional to dilute the biological sample tested.

The term “biological sample” encompasses any sample (fluid, tissue or cell) derived from an animal, typically from a mammal, from a lagomorph or from a rodent (rat, mouse, hamster, guinea pig), preferably from a human being. The biological sample is preferably a fluid, typically a fluid containing autoantibodies produced by said animal. The biological sample can be obtained directly from said animal or could be derived from a cell culture obtained from said animal. The biological samples are preferably chosen, for example, from blood, serum, plasma, cerebrospinal fluid (CSF), inner ear endolymph, inner ear perilymph, and a subfraction or a product derived therefrom. A preferred biological sample is a cerebrospinal fluid sample. The biological sample may have been subjected to a treatment such as a dilution in an acceptable vehicle. However, the present invention advantageously makes it possible to dispense with such a step.

The term “autoantibody” comprises any antibody and also any antibody fragment or portions such as Fab, F(ab′)2, Fv and scFv which bind to an epitope of a self protein.

The term “protein” covers any amino acid sequence, or set of amino acid sequences, comprising at least one antigen comprising at least one epitope. In the context of the present invention, the term “protein” typically covers a membrane protein or a membrane protein complex as defined previously.

The term “solubilized protein” denotes a protein having been exposed to a non-denaturing detergent, i.e., a non-ionic detergent (also identified as “mild detergent”), allowing the solubilization of said protein while at the same time preserving its conformation (three-dimensional structure) and its ability to bind to a support. This mild solubilization is the first step used for the purification of the membrane proteins and then subsequently their functional analysis.

The term “antigen” denotes a natural or synthetic macromolecule recognized by antibodies or cells of the immune system and capable of generating an immune response.

The term “epitope” (or “antigenic determinant”) denotes an immunoreactive sequence of amino acids, i.e., an amino acid sequence capable, by virtue of its structure, of specifically interacting with a particular antibody.

The same antigen may comprise several (identical or different) epitopes and thus induce varied immune responses. Sequential epitopes, corresponding to an amino acid sequence, and conformational epitopes, related to the authentic structure (spatial conformation or native three-dimensional conformation) of the protein, exist.

For the purposes of the invention, a protein can thus, for example, consist of a single-chain polypeptide; a set of polypeptides connected by covalent or non-covalent bonds; a protein portion, such as a subunit, a domain or a fragment; or a modified protein, for example modified by glycosylation (glycoprotein) or by combination with a lipid (lipoprotein). The term “protein” also covers the variants, derivatives and analogues of a particular protein.

The term “variant” or “derivative” is intended to mean any amino acid sequence which has been spontaneously modified (natural variant) or intentionally modified (artificial variant) compared with the specific amino acid sequence encoding the particular protein, such that the protein variant retains at least one of the endogenous functions of said particular protein, for example all of the endogenous functions of this particular protein (functionally equivalent protein). A variant can be obtained by addition or insertion, deletion and/or substitution of one or more amino acid residue(s) within the specific amino acid sequence encoding the particular protein.

The choice of the substitution amino acid can be decided on the basis of similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and/or of amphiphatic nature guaranteeing the preservation of a particular function (e.g., transport function or transport activity modulating function). Negatively charged amino acids include, for example, aspartic acid and glutamic acid; positively charged amino acids include, for example, lysine and arginine; and uncharged amino acids having a similar hydrophilic nature include, for example, leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine. Conservative substitutions can also be made, for example according to Table 1 below. The amino acids identified in the same block in the second column and preferably belonging to the same line in the third column can, for example, substitute for one another.

TABLE 1 ALIPHATIC Non-polar G A P I L V Polar-Uncharged C S T M N Q Polar-charged D E K R AROMATIC H F W Y

The term “analogue” is intended to mean any mimetic which is a naturally existing or artificially produced chemical compound which has at least one of the endogenous functions of the protein that it mimics, for example all of the endogenous functions of the protein that it mimics (functionally equivalent protein).

Generally, an epitope consists of a sequence of at least 3 to 4 amino acids, and more commonly of at least 5 to 6 amino acids. The epitopes may also consist of a sequence of approximately 7 to 8 amino acids, of even of approximately 10 amino acids. In the case of a sequential epitope, the amino acids are consecutive within the linear amino acid sequence of the protein. Conversely, as explained previously, the amino acids of a conformational epitope are not necessarily all consecutive within the linear amino acid sequence of the protein, the conformational epitope depending on the structure of the protein. In the context of the present invention, a particular epitope of a given protein denotes a (linear or conformational) particular amino acid sequence and also the immunological equivalents of the latter.

In one particular embodiment, the invention relates to a protein chip for detecting antibodies, typically autoantibodies, in a biological sample, said chip comprising at least one solubilized protein of interest (preferably a protein having retained its three-dimensional structure), typically at least one solubilized membrane protein of interest chosen, for example, from an ion channel protein, a transporter protein, and a membrane receptor protein which regulates the activity of an ion channel protein or of a transporter protein, said protein being optionally, when it corresponds to a main subunit, associated with one or more auxiliary subunits, and said chip being in the form of a support for detection of the complexes formed between antibodies and epitopes specific for said protein, the latter being optionally associated with its auxiliary subunit(s).

The term “ion channel protein” denotes a membrane protein which allows the passive passage of one or more ions. Ion channel proteins capable of being bound to the surface of the protein chip according to the invention can be chosen, for example, from ion channels and channel receptors.

Examples of ion channels are potassium channels (intracellular calcium-activated potassium channel, inwardly rectifying potassium channel, two-P-domain potassium channel, etc.), calcium channels, sodium channels and chloride channels. The ion channel may be a voltage gated channel.

Examples of channel receptors are the acetylcholine receptor, the glutamate receptor, the γ-aminobutyric acid (GABA) receptor and the adenosine-5′-triphosphate (ATP) receptor.

The term “transporter protein” denotes a protein intrinsic to the lipid cell membrane which allows the selective passage of water, of ions and/or of metabolites. Transporter proteins capable of being bound to the surface of the protein chip according to the invention can be chosen from, for example, aquaporins, connexins, Na+/K+ ATPases (or Na+/K+ pumps), H+/K+ ATPases (or H+/K+ pumps), Na+/H+ exchangers, Na+/Ca++ exchangers, and Na—K—Cl cotransporters.

The expression “membrane receptor protein which regulates the activity of an ion channel protein or of a transporter protein” or “regulatory membrane receptor protein” denotes any membrane protein, optionally associated with an ion channel protein and/or with a transporter protein, which regulates at least one activity of said proteins. Regulatory proteins of membrane receptor type which are capable of being bound to the surface of the protein chip according to the invention can be chosen, for example, from seven-transmembrane domains, G protein-coupled receptors and receptors of tyrosine kinase type.

As indicated above, said at least one protein of interest deposited on the support typically corresponds to a single-stranded protein (peptide) in its three-dimensional configuration, to the main subunit of a multistrand protein (polypeptide) in its natural configuration, i.e., three-dimensional configuration, or else to one of the main subunits of such a multistrand protein. This protein of interest may, however, correspond to or be associated with one or more auxiliary subunits of a set of functionally associated proteins (protein complex).

The term “auxiliary subunit” denotes the secondary parts of a protein or a protein complex, i.e., the parts other than the main subunit(s). The auxiliary subunit may be a membrane or perimembrane subunit. The auxiliary subunits of a protein often make it possible to regulate the activity (i.e., at least one of the functions) or a property (i.e., a functional characteristic) of said protein. In the case of an ion channel or of a transporter protein, for example, the main subunit generally allows the passage of ions or of metabolites, while the auxiliary subunits regulate this passage. Chaperone proteins and ionotropic receptors (“ionophores”) are examples of auxiliary subunits of a protein complex.

Preferably, the protein of interest is expressed in the cells of the nervous system of an animal, typically of a mammal, of a rodent (rat, mouse, hamster, guinea pig) or of a lagomorph, preferably of a human being. The protein of interest is thus preferably a human protein. It may also be a protein originating from an animal other than a human being, such as those previously identified. Preferably, such an animal protein has a sequence sufficiently close to that of the corresponding human isoform to enable the recognition, by the antibodies of the immune system of a human being, of at least one conformational epitope of said animal protein. Typically, the animal protein of interest exhibits a homology between its linear sequence and that of the human isoform of at least 50%, preferably of at least 75%, even more preferably of at least 95%.

The protein of interest is typically a human membrane protein selected from a pentameric receptor (or “cys-loop” receptor), an ATP receptor, an ionotropic glutamate receptor, a voltage gated channel, for example a voltage gated calcium, sodium or potassium channel, an intracellular calcium-activated potassium channel, an inwardly rectifying potassium channel, a two-P-domain potassium channel, a cyclic nucleotide-activatable ion channel, a TRP

(Transient Receptor Potential) ion channel, a chloride channel, an acid-sensitive cation channel, a sodium channel auxiliary subunit, and a calcium channel auxiliary subunit.

As previously indicated, the protein of interest may also be a rodent protein, typically a mouse or rat protein. Membrane proteins of interest originating from rodents can, for example, be chosen from a rat sodium channel, a rat calcium channel and a rat glutamate channel.

In the context of the present invention, the protein of interest, typically the membrane protein of interest, is preferably associated (or suspected of being associated) with a human pathological condition responsible for the appearance of autoantibodies.

In one particular embodiment of the invention, the protein of interest, typically the membrane protein of interest, is advantageously associated (or suspected of being associated) with an autoimmune (or presumed autoimmune) disease or pathological condition affecting the nervous system (neurological disease).

Examples of autoimmune pathological conditions affecting the nervous system comprise encephalopathies associated with neuronal excitability disorders, typically with neuronal receptor excitability disorders, and include, without being limited thereto, acquired epileptic syndromes, encephalitis, paraneoplastic neurological syndromes (PNSs), and non-paraneoplastic neurological syndromes with autoantibodies. Examples of convulsive or non-convulsive antoimmune (or presumed autoimmune) encephalopathies associated with neuronal excitability disorders are limbic encephalitis (which involves autoantibodies directed against proteins associated with voltage gated potassium channels or autoantibodies directly directed against these channels), Rasmussen encephalitis (associated with antibodies directed against glutamate receptors and acetylcholine receptors), post-infectious encephalitis, anti-NMDA receptor (anti-NMDAR) antibody encephalitis, Morvan's syndrome, Devic's disease or Devic's neuromyelitis optica (which involves autoantibodies directed against aquaporin-4), opsoclonus myoclonus syndromes in children (of post-infectious or paraneoplastic origin), encephalomyelitis, acquired epileptic syndromes, and paraneoplastic neurological syndromes (PNSs). Preferentially, the encephalopathy is an attack on the central nervous system which may be associated with neuronal excitability disorders of (demonstrated or suspected) autoimmune origin and of post-infectious or paraneoplastic origin.

In another particular embodiment of the invention, the protein of interest, typically the membrane protein of interest, is a protein associated (or suspected of being associated) with a peripheral neurological syndrome (possibly associated with an encephalopathy) of (demonstrated or suspected) autoimmune origin, and typically of post-infectious or paraneoplastic origin. The peripheral neurological syndromes may be responsible for muscle paralysis of pre- or post-synaptic origin. Isaacs' syndrome is an example of a peripheral neurological syndrome.

In another particular embodiment of the invention, the protein of interest, typically the membrane protein of interest, is a protein associated (or suspected of being associated) with a neurosensory disease (possibly associated with sensory receptor excitability disorders), for example with an inner ear disease, such as Ménière's disease or acquired or presumed congenital deafness.

A particular chip according to the invention comprises at least one membrane protein of interest, typically several membrane proteins of interest, said protein being encoded by a human cDNA sequence chosen from the nucleotide sequences identified in Table 2 below or corresponding to an amino acid sequence chosen from the amino acid sequences also identified in Table 2 below, preferably from the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 311.

TABLE 2 Amplified Protein Seq Ref (isoforms) Gene Complete name of the gene Nucleotide Seq Ref (isoforms) gene size (bp) and associated SEQ ID NOs “Cys-loop” receptors HTR3A serotonin 5-HT3 receptor A NM_000869 (Hs), NM_213621 1537 NP_000860 (Hs) (SEQ ID NO: 1), subunit (Hs), NM_013561 (Mm), NP_998786 (Hs) (SEQ ID NO: 2), NM_001099644 (Mm), NP_038589 (Mm), NP_001093114 NM_024394 (Rn) (Mm), NP_077370 (Rn) HTR3B serotonin 5-HT3 receptor B NM_006028 (Hs), NM_020274 1333 NP_006019 (Hs) (SEQ ID NO: 3), subunit (Mm), NM_022189 (Rn) NP_064670 (Mm), NP_071525 (Rn) HTR3C serotonin 5-HT3 receptor C NM_130770 1348 NP_570126 (Hs) (SEQ ID NO: 4) subunit HTR3D serotonin 5-HT3 receptor D NM_182537 844 NP_872343 (Hs) (SEQ ID NO: 5) subunit HTR3E serotonin 5-HT3 receptor E NM_182589 1420 NP_872395 (Hs) (SEQ ID NO: 6) subunit CHRNA1 nicotinic acetylcholine receptor NM_001039523 (Hs), 1450 NP_001034612 (Hs) (SEQ ID NO: 7), α1 subunit NM_007389 (Mm), NM_024485 NP_031415 (Mm), NP_077811 (Rn) (Rn) CHRNA2 nicotinic acetylcholine receptor NM_000742 (Hs), NM_144803 1591 NP_000733 (Hs) (SEQ ID NO: 8), α2 subunit (Mm), NM_133420 (Rn) NP_659052 (Mm), NP_596911 (Rn) CHRNA3 nicotinic acetylcholine receptor NM_000743 (Hs), NM_145129 1519 NP_000734 (Hs) (SEQ ID NO: 9), α3 subunit (Mm), NM_052805 (Rn) NP_660111 (Mm), NP_434692 (Rn) CHRNA4 nicotinic acetylcholine receptor NM_000744 (Hs), NM_015730 1885 NP_000735 (Hs) (SEQ ID NO: 10), α4 subunit (Mm), NM_024354 (Rn) NP_056545 (Mm), NP_077330 (Rn) CHRNA5 nicotinic acetylcholine receptor NM_000745 (Hs), NM_176844 1407 NP_000736 (Hs) (SEQ ID NO: 11), α5 subunit (Mm), NM_017078 (Rn) NP_789814 (Mm), NP_058774 (Rn) CHRNA6 nicotinic acetylcholine receptor NM_004198 (Hs), NM_021369 1489 NP_004189 (Hs) (SEQ ID NO: 12), α6 subunit (Mm), NM_057184 (Rn) NP_067344 (Mm), NP_476532 (Rn) CHRNA7 nicotinic acetylcholine receptor NM_000746 (Hs), NM_007390 1513 NP_000737 (Hs) (SEQ ID NO: 13), α7 subunit (Mm), NM_012832 (Rn) NP_031416 (Mm), NP_036964 (Rn) CHRNA9 nicotinic acetylcholine receptor NM_017581 (Hs), 1444 NP_060051 (Hs) (SEQ ID NO: 14), α9 subunit NM_001081104 (Mm), NP_001074573 (Mm), NP_075219 NM_022930 (Rn) (Rn) CHRNA10 nicotinic acetylcholine receptor NM_020402 (Hs), 1354 NP_065135 (Hs) (SEQ ID NO: 15), α10 subunit NM_001081424 (Mm), NP_001074893 (Mm), NP_072161 NM_022639 (Rn) (Rn) CHRNB1 nicotinic acetylcholine receptor NM_000747 (Hs), NM_009601 1510 NP_000738 (Hs) (SEQ ID NO: 16), β1 subunit (Mm), NM_012528 (Rn) NP_033731 (Mm), NP_036660 (Rn) CHRNB2 nicotinic acetylcholine receptor NM_000748 (Hs), NM_009602 1510 NP_000739 (Hs) (SEQ ID NO: 17), β2 subunit (Mm), NM_019297 (Rn) NP_033732 (Mm), NP_062170 (Rn) CHRNB3 nicotinic acetylcholine receptor NM_000749 (Hs), NM_173212 1381 NP_000740 (Hs) (SEQ ID NO: 18), β3 subunit (Mm), NM_133597 (Rn) NP_775304 (Mm), NP_598281 (Rn) CHRNB4 nicotinic acetylcholine receptor NM_000750 (Hs), NM_148944 1501 NP_000741 (Hs) (SEQ ID NO: 19), β4 subunit (Mm), NM_052806 (Rn) NP_683746 (Mm), NP_434693 (Rn) CHRND nicotinic acetylcholine receptor NM_000751 (Hs), NM_021600 1555 NP_000742 (Hs) (SEQ ID NO: 20), δ subunit (Mm), NM_019298 (Rn) NP_067611 (Mm), NP_062171 (Rn) CHRNE nicotinic acetylcholine receptor NM_000080 (Hs), NM_009603 1483 NP_000071 (Hs) (SEQ ID NO: 21), ε subunit (Mm), NM_017194 (Rn) NP_033733 (Mm), NP_058890 (Rn) CHRNG nicotinic acetylcholine receptor NM_005199 (Hs), NM_009604 1558 NP_005190 (Hs) (SEQ ID NO: 22), γ subunit (Mm), NM_019145 (Rn) NP_033734 (Mm), NP_062018 (Rn) GABRA1 GABA receptor α1 subunit NM_000806 (Hs), NM_010250 1375 NP_000797 (Hs) (SEQ ID NO: 23), (Mm), NM_183326 (Rn) NP_034380 (Mm), NP_899155 (Rn) GABRA2 GABA receptor α2 subunit NM_000807 (Hs), NM_008066 1360 NP_000798 (Hs) (SEQ ID NO: 24), (Mm) NP_032092 (Mm) GABRA3 GABA receptor α3 subunit NM_000808 (Hs), NM_008067 1483 NP_000799 (Hs) (SEQ ID NO: 25), (Mm), NM_017069 (Rn) NP_032093 (Mm), NP_058765 (Rn) GABRA4 GABA receptor α4 subunit NM_000809 (Hs), NM_010251 1666 NP_000800 (Hs) (SEQ ID NO: 26), (Mm), NM_080587 (Rn) NP_034381 (Mm), NP_542154 (Rn) GABRA5 GABA receptor α5 subunit NM_000810 (Hs), NM_176942 1390 NP_000801 (Hs) (SEQ ID NO: 27), (Mm), NM_017295 (Rn) NP_795916 (Mm), NP_058991 (Rn) GABRA6 GABA receptor α6 subunit NM_000811 (Hs), 1363 NP_000802 (Hs) (SEQ ID NO: 28), NM_001099641 (Mm), NP_001093111 (Mm), NP_068613 NM_021841 (Rn) (Rn) GABRB1 GABA receptor β1 subunit NM_000812 (Hs), NM_008069 1429 NP_000803 (Hs) (SEQ ID NO: 29), (Mm), NM_012956 (Rn) NP_032095 (Mm), NP_037088 (Rn) GABRB2 GABA receptor β2 subunit NM_021911 (Hs), NM_000813 1543 NP_068711 (Hs) (SEQ ID NO: 30), (Hs), NM_008070 (Mm), NP_000804 (Hs) (SEQ ID NO: 31), NM_012957 (Rn) NP_032096 (Mm), NP_037089 (Rn) GABRB3 GABA receptor β3 subunit NM_000814 (Hs), NM_008071 1426 NP_000805 (Hs) (SEQ ID NO: 32), (Mm), NM_017065 (Rn) NP_032097 (Mm), NP_058761 (Rn) GABRD GABA receptor δ subunit NM_000815 (Hs), NM_008072 1360 NP_000806 (Hs) (SEQ ID NO: 33), (Mm), NM_017289 (Rn) NP_032098 (Mm), NP_058985 (Rn) GABRE* GABA receptor ε subunit * NM_004961 (Hs), NM_017369 1525 NP_004952 (Hs) (SEQ ID NO: 34), (Mm), NM_023091 (Rn) NP_059065 (Mm), NP_075579 (Rn) GABRG1 GABA receptor γ1 subunit NM_173536 (Hs), NM_010252 1399 NP_775807 (Hs) (SEQ ID NO: 35), (Mm), NM_080586 (Rn) NP_034382 (Mm), NP_542153 (Rn) GABRG2 GABA receptor γ2 subunit NM_198904 (Hs), NM_008073 1432 NP_944494 (Hs) (SEQ ID NO: 36), (Mm), NM_183327 (Rn) NP_032099 (Mm), NP_899156 (Rn) GABRG3 GABA receptor γ3 subunit NM_033223 (Hs), NM_008074 1405 NP_150092 (Hs) (SEQ ID NO: 37), (Mm), NM_024370 (Rn) NP_032100 (Mm), NP_077346 (Rn) GABRP GABA receptor π subunit NM_014211 (Hs), NM_146017 1183 NP_055026 (Hs) (SEQ ID NO: 38), (Mm), NM_031029 (Rn) NP_666129 (Mm), NP_112291 (Rn) GABRR1 GABA receptor ρ1 subunit NM_002042 (Hs), NM_008075 1322 NP_002033 (Hs) (SEQ ID NO: 39), (Mm), NM_017291 (Rn) NP_032101 (Mm), NP_058987 (Rn) GABRR2 GABA receptor ρ2 subunit NM_002043 (Hs), NM_008076 1474 NP_002034 (Hs) (SEQ ID NO: 40), (Mm), NM_017292 (Rn) NP_032102 (Mm), NP_058988 (Rn) GABRR3 GABA receptor ρ3 subunit NM_001105580 (Hs), 1405 NP_001099050 (Hs) (SEQ ID NO: 41), NM_001081190 (Mm), NP_001074659 (Mm), NP_620252 NM_138897 (Rn) (Rn) GABRQ GABA receptor θ subunit NM_018558 (Hs), NM_020488 1183 NP_061028 (Hs) (SEQ ID NO: 42), (Mm), NM_031733 (Rn) NP_065234 (Mm), NP_113921 (Rn) GLRA1 glycine receptor α1 subunit NM_000171 (Hs), NM_020492 1354 NP_000162 (Hs) (SEQ ID NO: 43), (Mm), NM_013133 (Rn) NP_065238 (Mm), NP_037265 (Rn) GLRA2 glycine receptor α2 subunit NM_002063 (Hs), NM_183427 1363 NP_002054 (Hs) (SEQ ID NO: 44), (Mm), NM_012568 (Rn) NP_906272 (Mm), NP_036700 (Rn) GLRA3 glycine receptor α3 subunit NM_006529 (Hs), NM_080438 1396 NP_006520 (Hs) (SEQ ID NO: 45), (Mm), NM_053724 (Rn) NP_536686 (Mm), NP_446176 (Rn) GLRA4 glycine receptor α4 subunit NM_001024452.2 (Hs), 1135 NP_001019623 (Hs) (SEQ ID NO: 46), NM_010297 (Mm) NP_034427 (Mm), XP_346351.2 (Rn) GLRB glycine receptor β subunit NM_000824 (Hs), NM_010298 1498 NP_000815 (Hs) (SEQ ID NO: 47), (Mm), NM_053296 (Rn) NP_034428 (Mm), NP_445748 (Rn) ATP receptors P2RX1 ATP receptor P2X1 subunit NM_002558 (Hs), NM_008771 1201 NP_002549 (Hs) (SEQ ID NO: 48), (Mm), NM_012997 (Rn), NP_032797 (Mm), NP_037129 (Rn), AF231010 (Rn) Q9JIF8 (Rn) P2RX2 ATP receptor P2X2 subunit AF190824 (Hs), AF190823 (Hs), 1417 AAF19173 (Hs) (SEQ ID NO: 49), Isoform A AF190825 (Hs), NM_170682 NP_733782 (Hs) (SEQ ID NO: 50), (Hs), NM_153400 (Mm), AAF19171 (Hs) (SEQ ID NO: 51), NM_053656 (Rn) AAF19172 (Hs) (SEQ ID NO: 52), NP_700449 (Mm), NP_446108 (Rn) P2RX3 ATP receptor P2X3 subunit NM_002559 (Hs), NM_145526 1198 NP_002550 (Hs) (SEQ ID NO: 53), (Mm), NM_031075 (Rn) NP_663501 (Mm), NP_112337 (Rn) P2RX4 ATP receptor P2X4 subunit NM_002560 (Hs), NM_011026 1168 NP_002551 (Hs) (SEQ ID NO: 54), (Mm), NM_031594 (Rn) NP_035156 (Mm), NP_113782 (Rn) P2RX5 ATP receptor P2X5 subunit NM_002561 (Hs), NM_033321 1270 NP_002552 (Hs) (SEQ ID NO: 55), isoform A (Mm), NM_080780 (Rn) NP_201578 (Mm), NP_542958 (Rn) P2RXL1 ATP receptor P2X6 subunit (aka NM_005446 (Hs), NM_011028 1330 NP_005437 (Hs) (SEQ ID NO: 56), P2XM, and P2X-like 1) (Mm), NM_012721 (Rn) NP_035158 (Mm), NP_036853 (Rn) P2RX7 ATP receptor P2X7 subunit NM_002562 (Hs), NM_011027 1792 NP_002553 (Hs) (SEQ ID NO: 57), (Mm), FJ436444 (Mm), NP_035157 (Mm), ACR61395 (Mm), NM_019256 (Rn), FJ436445 ACR61396 (Rn), NP_062129 (Rn) (Rn) Ionotropic glutamate receptors Orphan glutamate receptor δ1 subunit NM_017551 (Hs), NM_008166 3031 NP_060021 (Hs) (SEQ ID NO: 58), GRID1 (Mm), NM_024378 (Rn) NP_032192 (Mm), NP_077354 (Rn) Orphan glutamate receptor δ2 subunit NM_001510 (Hs), NM_008167 3025 NP_001501 (Hs) (SEQ ID NO: 59), GRID2 (Mm), NM_024379 (Rn) NP_032193 (Mm), NP_077355 (Rn) Kainate glutamate receptor KA1 subunit NM_014619 (Hs), NM_175481 1875 NP_055434 (Hs) (SEQ ID NO: 60), GRIK4 (aka EAA1) (Mm), NM_012572 (Rn) NP_780690 (Mm), NP_036704 (Rn) Kainate glutamate receptor KA2 subunit NM_002088 (Hs), NM_008168 2947 NP_002079 (Hs) (SEQ ID NO: 61), GRIK5 (aka EAA2) (Mm), NM_031508 (Rn) NP_032194 (Mm), NP_113696 (Rn) NMDA glutamate receptor NMDAR1 NM_007327 (Hs), NM_008169 2665 NP_015566 (Hs) (SEQ ID NO: 62), GRIN1 subunit (Mm), NM_017010 (Rn) NP_032195 (Mm), NP_058706 (Rn) NMDA glutamate receptor NMDAR2A NM_000833 (Hs), NM_008170 4396 NP_000824 (Hs) (SEQ ID NO: 63), GRIN2A subunit (Mm), NM_012573 (Rn) NP_032196 (Mm), NP_036705 (Rn) NMDA glutamate receptor NMDAR2B NM_000834 (Hs), NM_008171 4459 NP_000825 (Hs) (SEQ ID NO: 64), GRIN2B subunit (Mm), NM_012574 (Rn) NP_032197 (Mm), NP_036706 (Rn) NMDA glutamate receptor NMDAR2C NM_000835 (Hs), NM_010350 3706 NP_000826 (Hs) (SEQ ID NO: 65), GRIN2C subunit (Mm), NM_012575 (Rn) NP_034480 (Mm), NP_036707 (Rn) NMDA glutamate receptor NMDAR2D NM_000836 (Hs), NM_008172 4015 NP_000827 (Hs) (SEQ ID NO: 66), GRIN2D subunit (Mm), NM_022797 (Rn) NP_032198 (Mm), NP_073634 (Rn) NMDA glutamate receptor NMDAR3A NM_133445 (Hs), 3352 NP_597702 (Hs) (SEQ ID NO: 67), GRIN3A subunit NM_001033351 (Mm) NP_001028523 (Mm) NMDA glutamate receptor NMDAR3B NM_138690 (Hs), NM_130455 3133 NP_619635 (Hs) (SEQ ID NO: 68), GRIN3B subunit (aka χ-2) (Mm), NM_133308 (Rn) NP_569722 (Mm), NP_579842 (Rn) AMPA glutamate receptor GLUR1 NM_000827 (Hs), 2725 NP_000818 (Hs) (SEQ ID NO: 69), GRIA1 subunit (aka KR4, HBGR1, NM_001114183 (Hs), NP_001107655 (Hs) (SEQ ID NO: 70), GluHI) NM_008165 (Mm), NP_001106796 (Mm), NP_032191 NM_001113325 (Mm), (Mm), NP_113796 (Rn) NM_031608 (Rn) AMPA glutamate receptor GLUR2 NM_001083619 (Hs), 2656 NP_000817 (Hs) (SEQ ID NO: 71), GRIA2 subunit NM_000826 (Hs), NM_013540 NP_001077088 (Hs) (SEQ ID NO: 72), (Mm), NM_001083806 (Mm), NP_001077275 (Mm), NP_001034284 NM_001039195 (Mm), (Mm), NP_038568 (Mm), NP_058957 NM_017261 (Rn), (Rn), NP_001077280 (Rn) NM_001083811 (Rn) AMPA glutamate receptor GLUR3 NM_007325 (Hs), NM_000828 2686 NP_015564 (Hs) (SEQ ID NO: 73), GRIA3 subunit (aka GLURC) (Hs), NM_016886 (Mm), NP_000819 (Hs) (SEQ ID NO: 74), NM_001112742 (Rn), NP_058582 (Mm), NP_116785 (Rn), NM_032990 (Rn) NP_001106213 (Rn) AMPA glutamate receptor GluR4 NM_001077243 (Hs), 2713 NP_000820 (Hs) (SEQ ID NO: 75), GRIA4 subunit NM_000829 (Hs), NP_001070711 (Hs) (SEQ ID NO: 76), NM_001077243 (Hs), NP_000820 (Hs), NP_001070711 (Hs), NM_000829 (Hs), NP_062665 (Mm), NP_001106651 NM_001113180 (Mm), (Mm), NP_058959 (Rn), NM_019691 (Mm), NP_001106655 (Rn) NM_001113184 (Rn), NM_017263 (Rn) Kainate glutamate receptor GLUR5 NM_175611 (Hs), NM_010348 2761 NP_783300 (Hs) (SEQ ID NO: 77), GRIK1 subunit (aka EEA3) (Mm), NM_017241 (Rn) NP_034478 (Mm), NP_058937 (Rn) Kainate glutamate receptor GLUR6 NM_021956 (Hs), NM_010349 2731 NP_068775 (Hs) (SEQ ID NO: 78), GRIK2 subunit (aka EAA4 aka β2) (Mm), NM_019309 (Rn) NP_034479 (Mm), NP_062182 (Rn) Kainate glutamate receptor GLUR7 NM_000831 (Hs), 2764 NP_000822 (Hs) (SEQ ID NO: 79), GRIK3 subunit (aka EAA5) NM_001081097 (Mm), NP_001074566 (Mm), NP_001106187 NM_001112716 (Rn) (Rn) Voltage gated sodium channels SCN1A voltage gated sodium channel NM_006920 (Hs), NM_018733 5998 NP_008851 (Hs) (SEQ ID NO: 80), type I a subunit (Mm), NM_030875 (Rn) NP_061203 (Mm) (SEQ ID NO: 81), NP_110502 (Rn) (SEQ ID NO: 82) SCN2A voltage gated sodium channel NM_001040143 (Hs), 6019 NP_001035232 (Hs) (SEQ ID NO: 83), type II a subunit NM_001040142 (Hs), NP_001035233 (Hs) (SEQ ID NO: 84), NM_001099298 (Mm), NP_001092768 (Mm) (SEQ ID NO: NM_012647 (Rn) 85), NP_036779 (Rn) (SEQ ID NO: 86) SCN3A voltage gated sodium channel NM_006922 (Hs), NM_018732 6004 NP_008853 (Hs) (SEQ ID NO: 87), type III a subunit (Mm), NM_013119 (Rn) NP_061202 (Mm) (SEQ ID NO: 88), NP_037251 (Rn) (SEQ ID NO: 89) SCN4A voltage gated sodium channel NM_000334 (Hs), NM_133199 5512 NP_000325 (Hs) (SEQ ID NO: 90), type IV a subunit (Mm), NM_013178 (Rn) NP_573462 (Mm) (SEQ ID NO: 91), NP_037310 (Rn) (SEQ ID NO: 92) SCN5A voltage gated sodium channel NM_001099404 (Hs), 6055 NP_001092874 (Hs) (SEQ ID NO: 93), type V a subunit NM_000335 (Hs), NM_198056 NP_001092875 (Hs) (SEQ ID NO: 94), (Hs), NM_001099405 (Hs), NP000326 (Hs) (SEQ ID NO: 95), NM_021544 (Mm), NM_013125 NP_932173 (Hs) (SEQ ID NO: 96), (Rn) NP_067519 (Mm) (SEQ ID NO: 97), NP_037257 (Rn) (SEQ ID NO: 98) SCN8A voltage gated sodium channel NM_014191 (Hs), 5944 NP_055006 (Hs) (SEQ ID NO: 99), type VIII a subunit NM_001077499 (Mm), NP_001070967 (Mm) (SEQ ID NO: NM_019266 (Rn) 100), NP_062139 (Rn) (SEQ ID NO: 101) SCN9A voltage gated sodium channel NM_002977 (Hs), NM_018852 5935 NP_002968 (Hs) (SEQ ID NO: 102), type IX a subunit (Mm), NM_133289 (Rn) NP_061340 (Mm) (SEQ ID NO: 103), NP_579823 (Rn) (SEQ ID NO: 104) SCN10A voltage gated sodium channel NM_006514 (Hs), NM_009134 5872 NP_006505 (Hs) (SEQ ID NO: 105), type X a subunit (Mm), NM_017247 (Rn) NP_033160 (Mm) (SEQ ID NO: 106), NP_058943 (Rn) (SEQ ID NO: 107) SCN11A voltage gated sodium channel NM_014139 (Hs), NM_011887 5377 NP_054858 (Hs) (SEQ ID NO: 108), type XI a subunit (Mm), NM_019265 (Rn) NP_036017 (Mm) (SEQ ID NO: 109), NP_062138 (Rn) (SEQ ID NO: 110) Voltage gated calcium channels CACNA1S voltage gated calcium channel NM_000069 (Hs), 5626 NP_000060 (Hs) (SEQ ID NO: 111), L-type a1S subunit NM_001081023 (Mm), NP_001074492 (Mm) (SEQ ID NO: NM_053873 (Rn) 112), NP_446325 (Rn) (SEQ ID NO: 113) CACNA1C voltage gated calcium channel NM_000719 (Hs), NM_009781 6418 NP_000710 (Hs) (SEQ ID NO: 114), L-type a1C subunit (Mm), NM_012517 (Rn) NP_033911 (Mm) (SEQ ID NO: 115), NP_036649 (Rn) (SEQ ID NO: 116) CACNA1D voltage gated calcium channel NM_000720 (Hs), NM_028981 6550 NP_000711 (Hs) (SEQ ID NO: 117), L-type a1D subunit (Mm), NM_017298 (Rn) NP_083257 (Mm) (SEQ ID NO: 118), NP_058994 (Rn) (SEQ ID NO: 119) CACNA1F voltage gated calcium channel NM_005183 (Hs), NM_019582 5938 NP_005174 (Hs) (SEQ ID NO: 120), L-type a1F subunit (Mm), NM_053701 (Rn) NP_062528 (Mm) (SEQ ID NO: 121), NP_446153 (Rn) (SEQ ID NO: 122) CACNA1A voltage gated calcium channel NM_023035 (Hs), 6802 NP_075461 (Hs) (SEQ ID NO: 123), P/Q-type a1A subunit NM_000068.3 (Hs), NP_000059 (Hs) (SEQ ID NO: 124), NM_007578 (Mm), NM_012918 NP_031604 (Mm) (SEQ ID NO: 125), (Rn) NP_037050 (Rn) (SEQ ID NO: 126) CACNA1B voltage gated calcium channel NM_000718 (Hs), 7021 NP_000709 (Hs) (SEQ ID NO: 127), N-type a1B subunit NM_001042528 (Mm), NP_001035993 (Mm) (SEQ ID NO: NM_147141 (Rn) 128), NP_671482 (Rn) (SEQ ID NO: 129) CACNA1E voltage gated calcium channel NM_000721 (Hs), NM_009782 6814 NP_000712 (Hs) (SEQ ID NO: 130), R-type a1E subunit (Mm), NM_019294 (Rn) NP_033912 (Mm) (SEQ ID NO: 131), NP_062167 (Rn) (SEQ ID NO: 132) CACNA1G voltage gated calcium channel NM_198378 (Hs), NM_198387 7135 NP_061496 (Hs) (SEQ ID NO: 133), T-type a1G subunit (Hs), NM_198379 (Hs), NP_938192 (Hs) (SEQ ID NO: 134), NM_938190 (Hs), NM_009783 NP_938190 (Hs) (SEQ ID NO: 135), (Mm), NM_031601 (Rn) NP_938201 (Hs) (SEQ ID NO: 136), NP_938193 (Hs) (SEQ ID NO: 137), NP_033913 (Mm) (SEQ ID NO: 138), NP_113789 (Rn) (SEQ ID NO: 139) CACNA1H voltage gated calcium channel NM_021098 (Hs), NM_021415 7069 NP_066921 (Hs) (SEQ ID NO: 140), T-type a1H subunit (Mm), NM_153814 (Rn) NP_067390 (Mm) (SEQ ID NO: 141), NP_722521 (Rn) (SEQ ID NO: 142) CACNA1I voltage gated calcium channel NM_001003406 (Hs), 6673 NP_066919 (Hs) (SEQ ID NO: 143), T-type a1I subunit NM_021096 (Hs), NP_001003406 (Hs) (SEQ ID NO: NM_001044308 (Mm), 144), NP_001037773 (Mm) (SEQ ID NM_020084 (Rn) NO: 145), NP_064469 (Rn) (SEQ ID NO: 146) Voltage gated potassium channels Shaker voltage gated Potassium channel NM_000217 (Hs), NM_010595 1492 NP_000208 (Hs) (SEQ ID NO: 147), KCNA1 Delayed Rectifier member 1 (Mm), NM_173095 (Rn) NP_034725 (Mm), NP_775118 (Rn) Shaker voltage gated Potassium channel NM_004974 (Hs), NM_008417 1504 NP_004965 (Hs) (SEQ ID NO: 148), KCNA2 Delayed Rectifier member 2 (Mm), NM_012970 (Rn) NP_032443 (Mm), NP_037102 (Rn) Shaker voltage gated Potassium channel NM_002232 (Hs), NM_008418 1729 NP_002223 (Hs) (SEQ ID NO: 149), KCNA3 Delayed Rectifier member 3 (Mm), NM_019270 (Rn) NP_032444 (Mm), NP_062143 (Rn) Shaker voltage gated Potassium channel NM_002233 (Hs), NM_021275 1963 NP_002224 (Hs) (SEQ ID NO: 150), KCNA4 A-Type, Fast inactivation (Mm), NM_012971 (Rn) NP_067250 (Mm), NP_037103 (Rn) member 4 Shaker voltage gated Potassium channel NM_002234 (Hs), NM_145983 1843 NP_002225 (Hs) (SEQ ID NO: 151), KCNA5 Delayed Rectifier member 5 (Mm), NM_012972 (Rn) NP_666095 (Mm), NP_037104 (Rn) Shaker voltage gated Potassium channel NM_002235 (Hs), NM_013568 1597 NP_002226 (Hs) (SEQ ID NO: 152), KCNA6 Delayed Rectifier member 6 (Mm), NM_023954 (Rn) NP_038596 (Mm), NP_076444 (Rn) Shaker voltage gated Potassium channel NM_031886 (Hs), NM_010596 1375 NP_114092 (Hs) (SEQ ID NO: 153), KCNA7 Delayed Rectifier member 7 (Mm), NM_001108914 (Rn) NP_034726 (Mm), NP_001102384 (Rn) Shaker voltage gated Potassium channel NM_005549 (Hs), 1537 NP_005540 (Hs) (SEQ ID NO: 154), KCNA10 Delayed Rectifier member 10 NM_001074609 (Mm), NP_001074609 (Mm), XP_227577 XM_227577 (Rn) (Rn) Shab voltage gated Potassium channel NM_004975 (Hs), NM_008420 2581 NP_004966 (Hs) (SEQ ID NO: 155), KCNB1 Delayed Rectifier Shab Related (Mm), NM_013186 (Rn) NP_032446 (Mm), NP_037318 (Rn) member 1 Shab voltage gated Potassium channel NM_004770 (Hs), 2737 NP_004761 (Hs) (SEQ ID NO: 156), KCNB2 Delayed Rectifier Shab Related NM_001098528 (Mm), NP_001091998 (Mm), NP_446452 member 2 NM_054000 (Rn) (Rn) Shaw voltage gated Potassium channel NM_004976 (Hs), NM_008421 1537 NP_004967 (Hs) (SEQ ID NO: 157), KCNC1 Delayed Rectifier Shaw Related (Mm), NM_001112739 (Mm), NP_032447 (Mm), NP_001106210 member 1 NM_139217.1 (Rn), (Mm), NP_631963 (Rn), NP_036988 NM_012856 (Rn) (Rn) Shaw voltage gated Potassium channel NM_153748 (Hs), NM_139136 1843 NP_631875 (Hs) (SEQ ID NO: 158), KCNC2 Delayed Rectifier Shaw Related (Hs), NM_139137 (Hs), NP_715624 (Hs) (SEQ ID NO: 159), member 2 NM_001025581 (Mm), NP_631874 (Hs) (SEQ ID NO: 160), NM_139217 (Rn), NM_139216 NP_001020752 (Mm), NP_631962 (Rn) (Rn), NP_631963 (Rn) Shaw voltage gated Potassium channel NM_004977 (Hs), NM_008422 2278 NP_004968 (Hs) (SEQ ID NO: 161), KCNC3 A-Type Shaw Related member 3 (Mm), NM_053997 (Rn) NP_032448 (Mm), NP_446449 (Rn) Shaw voltage gated Potassium channel NM_004978 (Hs), NM_145922 1912 NP_004969 (Hs) (SEQ ID NO: 162), KCNC4 A-Type Fast inactivation Shaw (Mm) NP_666034 (Mm), NP_001116248 Related member 4 (Rn) Shal voltage gated Potassium channel NM_004979 (Hs), NM_008423 1948 NP_004970 (Hs) (SEQ ID NO: 163), KCND1 A-Type Shal Related member 1 (Mm), NM_001105748 (Rn) NP_032449 (Mm), NP_001099218 (Rn) Shal voltage gated Potassium channel NM_012281 (Hs), NM_019697 1894 NP_036413 (Hs) (SEQ ID NO: 164), KCND2 A-Type Shal Related member 2 (Mm), NM_031730 (Rn) NP_062671 (Mm), NP_113918 (Rn) Shal voltage gated Potassium channel NM_172198 (Hs), NM_004980 1969 NP_004971 (Hs) (SEQ ID NO: 165), KCND3 A-Type Shal Related member 3 (Hs), NM_019931 (Mm), NP_751948 (Hs) (SEQ ID NO: 166), NM_031739 (Rn) NP_064315 (Mm), NP_113927 (Rn) Modifier voltage gated Potassium channel NM_002236 (Hs), NM_201531 1486 NP_002227 (Hs) (SEQ ID NO: 167), KCNF1 modifier of Kv2 (Mm), NM_001169104 (Rn) NP_963289 (Mm), NP_001162575 (Rn) Modifier voltage gated Potassium channel NM_002237 (Hs), 1546 NP_002228 (Hs) (SEQ ID NO: 168), KCNG1 modifier/silencer of Kv2 NM_001081134 (Mm), NP_001074603 (Mm), NP_001100015 NM_001106545 (Rn) (Rn) Modifier voltage gated Potassium channel NM_012283 (Hs), 1403 NP_036415.1 (Hs) (SEQ ID NO: 169), KCNG2 modifier/silencer of Kv2 NM_012283.1 (Hs), NP_036415 (Hs) (SEQ ID NO: 170), XM_140499 (Mm), XP_140499 (Mm), NP_001100842 NM_001107372 (Rn) (Rn) Modifier voltage gated Potassium channel NM_133329 (Hs), NM_172344 1315 NP_579875 (Hs) (SEQ ID NO: 171), KCNG3 modifier/silencer of Kv2 (Hs), NM_153512 (Mm), NP_758847 (Hs) (SEQ ID NO: 172), NM_001033957 (Rn), NP_705732 (Mm), NP_596917 (Rn), NM_133426 (Rn) NP_001029129 (Rn) Modifier voltage gated Potassium channel NM_172347 (Hs), NM_025734 1564 NP_758857 (Hs) (SEQ ID NO: 173), KCNG4 modifier/silencer of Kv2 (Mm), NM_001107435 (Rn) NP_080010 (Mm), NP_001100905 (Rn) KCNQ1 voltage gated Potassium channel NM_000218 (Hs), NM_181798 2032 NP_000209 (Hs) (SEQ ID NO: 174), Delayed Rectifier KQT-Like (Hs), NM_008434 (Mm), NP_032460 (Mm), NP_114462 (Rn) member 1 NM_032073 (Rn) KCNQ2 voltage gated Potassium channel NM_172107 (Hs), NM_010611 2620 NP_742105 (Hs) (SEQ ID NO: 175), Delayed Rectifier KQT-Like (Mm), NM_133322 (Rn) NP_034741 (Mm), NP_579856 (Rn) member 2 KCNQ3 voltage gated Potassium channel NM_004519 (Hs), NM_152923 2620 NP_004510 (Hs) (SEQ ID NO: 176), Delayed Rectifier KQT-Like (Mm), NM_031597 (Rn) NP_690887 (Mm), NP_113785 (Rn) member 3 KCNQ4 voltage gated Potassium channel NM_172163.1 (Hs), 2089 NP_751895 (Hs) (SEQ ID NO: 177), Delayed Rectifier KQT-Like NM_004700 (Hs), NP_004691 (Hs) (SEQ ID NO: 178), member 4 NM_001081142 (Mm), NP_001074611 (Mm), XP_233477 XM_233477 (Rn) (Rn) KCNQ5 voltage gated Potassium channel NM_019842 (Hs), NM_023872 2803 NP_062816 (Hs) (SEQ ID NO: 179), modifier/silencer of Kv2 (Mm), XM_001071249 (Rn) NP_076361 (Mm), XP_001071249 (Rn) KCNV1 voltage gated Potassium channel NM_014379 (Hs), NM_026200 1507 NP_055194 (Hs) (SEQ ID NO: 180), modifier/silencer of Kv2 (Mm), NM_021697 (Rn) NP_080476 (Mm), NP_067729 (Rn) KCNV2 voltage gated Potassium channel NM_133497 (Hs), NM_183179 1642 NP_598004 (Hs) (SEQ ID NO: 181), modifier/silencer of Kv2 (Mm), NM_001106370 (Rn) NP_899002 (Mm), NP_001099840 (Rn) KCNS1 voltage gated Potassium channel NM_002251 (Hs), NM_008435 1585 NP_002242 (Hs) (SEQ ID NO: 182), modifier/silencer of Kv2 (Mm), NM_053954 (Rn) NP_032461 (Mm), NP_446406 (Rn) KCNS2 voltage gated Potassium channel NM_020697 (Hs), NM_181317 1438 NP_065748 (Hs) (SEQ ID NO: 183), modifier/silencer of Kv2 (Mm), NM_023966 (Rn) NP_851834 (Mm), NP_076456 (Rn) KCNS3 voltage gated Potassium channel NM_002252 (Hs), NM_173417 1477 NP_002243 (Hs) (SEQ ID NO: 184), modifier/silencer of Kv2 (Mm), NM_031778 (Rn) NP_775593 (Mm), NP_113966 (Rn) KCNH1 voltage gated Potassium channel NM_172362 (Hs), NM_010600 2974 NP_758872 (Hs) (SEQ ID NO: 185), Delayed Rectifier Ether-A-Gogo (Mm), NM_031742 (Rn) NP_034730 (Mm), NP_113930 (Rn) (eag1) KCNH5 voltage gated Potassium channel NM_172375 (Hs), NM_172376 2971 NP_758963 (Hs) (SEQ ID NO: 186), Outward-Rectifying Ether-A- (Hs), NM_139318 (Hs), NP_758964 (Hs) (SEQ ID NO: 187), Gogo (eag2) NM_172805 (Mm), NM_133610 NP_647479 (Hs), (Rn) NP_766393 (Mm), NP_598294 (Rn) KCNH2 voltage gated Potassium channel NM_172056 (Hs), NM_000238 3484 NP_000229 (Hs) (SEQ ID NO: 188), Inwardly-Rectifying Ether-A- (Hs), NM_172057 (Hs), NP_742053 (Hs) (SEQ ID NO: 189), Gogo (erg1 or HERG) NM_013569 (Mm), NM_053949 NP_742054 (Hs) (SEQ ID NO: 190), (Rn) NP_038597 (Mm), NP_446401 (Rn) KCNH6 voltage gated Potassium channel NM_030779 (Hs), NM_173092 2989 NP_110406 (Hs) (SEQ ID NO: 191), Inwardly-Rectifying Ether-A- (Hs), NM_001037712 (Mm), NP_775115 (Hs) (SEQ ID NO: 192), Gogo (erg2) NM_053937 (Rn) NP_001032801 (Mm), NP_446389 (Rn) KCNH7 voltage gated Potassium channel NM_173162 (Hs), NM_033272 3595 NP_775185 (Hs) (SEQ ID NO: 193), Inwardly-Rectifying Ether-A- (Hs), NM_133207 (Mm), NP_150375 (Hs) (SEQ ID NO: 194), Gogo (erg3) NM_131912 (Rn) NP_573470 (Mm), NP_571987 (Rn) KCNH8 voltage gated Potassium channel NM_144633 (Hs), 3328 NP_653234 (Hs) (SEQ ID NO: 195), Inwardly-Rectifying Ether-A- NM_001031811 (Mm), NP_001026981 (Mm), NP_659563 Gogo (elk1) NM_145095 (Rn) (Rn) KCNH3 voltage gated Potassium channel NM_012284 (Hs), NM_010601 3259 NP_036416 (Hs) (SEQ ID NO: 196), Inwardly-Rectifying Ether-A- (Mm), NM_017108 (Rn) NP_034731 (Mm), NP_058804 (Rn) Gogo (elk2) KCNH4 voltage gated Potassium channel NM_012285 (Hs), 3058 NP_036417 (Hs) (SEQ ID NO: 197), Inwardly-Rectifying Ether-A- NM_001081194 (Mm), NP_001074663 (Mm), NP_446082 Gogo (elk1) NM_053630 (Rn) (Rn) Intracellular calcium-activated potassium channels KCNMA1 large conductance calcium- NM_001014797 (Hs), 3550 NP_001014797 (Hs) (SEQ ID NO: activated Potassium channel NM_010610 (Mm), NM_031828 198), NP_034740 (Mm), NP_114016 Slo1 (Rn) (Rn) KCNN1 small conductance calcium- NM_002248 (Hs), NM_032397 1636 NP_002239 (Hs) (SEQ ID NO: 199), activated Potassium channel (Mm), NM_019313 (Rn) NP_115773 (Mm), NP_062186 (Rn) SK1 KCNN2 small conductance calcium- NM_170775 (Hs), NM_021614 1744 NP_740721 (Hs) (SEQ ID NO: 200), activated Potassium channel (Hs), NM_080465 (Mm), NP_067627 (Hs) (SEQ ID NO: 201), SK2 NM_019314 (Rn) NP_536713 (Mm), NP_062187 (Rn) KCNN3 small conductance calcium- NM_002249 (Hs), NM_170782 2197 NP_740752 (Hs) (SEQ ID NO: 202), activated Potassium channel (Hs), NM_080466 (Mm), NP_002240 (Hs) (SEQ ID NO: 203), SK3 NM_019315 (Rn) NP_536714 (Mm), NP_062188 (Rn) KCNN4 intermediate conductance NM_002250 (Hs), NM_008433 1285 NP_002241 (Hs) (SEQ ID NO: 204), calcium-activated Potassium (Mm), NM_023021 (Rn) NP_032459 (Mm), NP_075410 (Rn) channel SK4 KCNT1 sodium activated Potassium NM_020822 (Hs), NM_175462 3775 NP_065873 (Hs) (SEQ ID NO: 205), channel Slo2.2 (Slack) (Mm), NM_021853 (Rn) NP_780671 (Mm), NP_068625 (Rn) KCNT2 sodium activated Potassium NM_198503 (Hs), 3409 NP_940905 (Hs) (SEQ ID NO: 206), channel Slo2.1 (Slick) NM_001081027 (Mm), NP_001074496 (Mm), NP_942057 NM_198762 (Rn) (Rn) KCNU1 sodium activated Potassium NM_001031836 (Hs), 3454 NP_001027006 (Hs) (SEQ ID NO: channel Slo3 NM_008432 (Mm) 207), NP_032458 (Mm) Inwardly rectifying potassium channels KCNJ1 Inwardly rectifying potassium NM_000220 (Hs), NM_153764 1180 NP_722448 (Hs) (SEQ ID NO: 208), channel Romk1 (Hs), NM_019659 (Mm), NP_000211 (Hs) (SEQ ID NO: 209), NM_017023 (Rn) NP_062633 (Mm), NP_058719 (Rn) KCNJ2 Inwardly rectifying potassium NM_000891 (Hs), NM_008425 1285 NP_000882 (Hs) (SEQ ID NO: 210), channel IRK1 (Mm), NM_017296 (Rn) NP_032451 (Mm), NP_058992 (Rn) KCNJ12 Inwardly rectifying potassium NM_021012 (Hs), NM_010603 1306 NP_066292 (Hs) (SEQ ID NO: 211), channel IRK2 (Mm), NM_053981 (Rn) NP_034733 (Mm), NP_446433 (Rn) KCNJ4 Inwardly rectifying potassium NM_152868 (Hs), NM_008427 1342 NP_690607 (Hs) (SEQ ID NO: 212), channel IRK3 (Mm), NM_053870 (Rn) NP_032453 (Mm), NP_446322 (Rn) KCNJ14 Inwardly rectifying potassium NM_170720 (Hs), NM_145963 1312 NP_733838 (Hs) (SEQ ID NO: 213), channel IRK4 (Mm), NM_170718 (Rn) NP_666075 (Mm), NP_733836 (Rn) KCNJ3 Inwardly rectifying potassium NM_002239 (Hs), NM_008426 1510 NP_002230 (Hs) (SEQ ID NO: 214), channel GIRK1 (Mm), NM_031610 (Rn) NP_032452 (Mm), NP_113798 (Rn) KCNJ6 Inwardly rectifying potassium NM_002240 (Hs), NM_010606 1273 NP_002231 (Hs) (SEQ ID NO: 215), channel GIRK2 (Mm), NM_001025590 (Mm), NP_001020756 (Mm), NP_001020755 NM_001025585 (Mm), (Mm), NP_034736 (Mm), NM_001025584 (Mm), NP_001020761 (Mm), NP_037324 NM_013192 (Rn) (Rn) KCNJ9 Inwardly rectifying potassium NM_004983 (Hs), NM_008429 1186 NP_004974 (Hs) (SEQ ID NO: 216), channel GIRK3 (Mm), NM_053834 (Rn) NP_032455 (Mm), NP_446286 (Rn) KCNJ5 Inwardly rectifying potassium NM_000890 (Hs), NM_010605 1264 NP_000881 (Hs) (SEQ ID NO: 217), channel GIRK4 (Mm), NM_017297 (Rn) NP_034735 (Mm), NP_058993 (Rn) KCNJ10 Inwardly rectifying potassium NM_002241 (Hs), 1144 NP_002232 (Hs) (SEQ ID NO: 218), channel BIR10 NM_001039484 (Mm), NP_001034573 (Mm), NP_113790 NM_031602 (Rn) (Rn) KCNJ15 Inwardly rectifying potassium NM_170736 (Hs), 1129 NP_733932 (Hs) (SEQ ID NO: 219), channel IRKK (Kir1.3) NM_001039057 (Mm), NP_001034145 (Mm), NP_001034146 NM_001039056 (Mm), (Mm), NP_579855 (Rn) NM_133321 (Rn) KCNJ16 Inwardly rectifying potassium NM_018658 (Hs), NM_010604 1261 NP_061128 (Hs) (SEQ ID NO: 220), channel BIR9 (Mm), NM_053314 (Rn) NP_034734 (Mm), NP_445766 (Rn) KCNJ8 ATP-sensitive potassium NM_004982 (Hs), NM_008428 1279 NP_004973 (Hs) (SEQ ID NO: 221), channel K-ATP1 (Mm), NM_017099 (Rn) NP_032454 (Mm), NP_058795 (Rn) KCNJ11 ATP-sensitive potassium NM_000525 (Hs), NM_010602 1177 NP_000516 (Hs) (SEQ ID NO: 222), channel BIR (Mm), NM_031358 (Rn) NP_034732 (Mm), NP_112648 (Rn) KCNJ13 ATP-sensitive potassium NM_002242 (Hs), NM_053608 1084 NP_002233 (Hs) (SEQ ID NO: 223), channel (Kir1.4) (Rn) NP_446060 (Rn) 2-P-domain potassium channels KCNK1 two pore domain potasium NM_002245 (Hs), NM_008430 1008 NP_002236 (Hs) (SEQ ID NO: 224), channel TWIK1 (Mm), NM_021688 (Rn) NP_032456 (Mm), NP_067720 (Rn) KCNK2 two pore domain potasium NM_001017424 (Hs), 1278 NP_001017424 (Hs) (SEQ ID NO: channel TREK1 NM_010607 (Mm), NM_172042 225), NP_034737 (Mm), NP_742038 (Rn) (Rn) KCNK3 two pore domain potasium NM_002246 (Hs), NM_010608 1182 NP_002237 (Hs) (SEQ ID NO: 226), channel TASK1 (Mm), NM_033376 (Rn) NP_034738 (Mm), NP_203694 (Rn) KCNK4 two pore domain potasium NM_033310 (Hs), NM_008431 1179 NP_201567 (Hs) (SEQ ID NO: 227), channel TRAAK (Mm), NM_053804 (Rn) NP_032457 (Mm), NP_446256 (Rn) KCNK5 two pore domain potasium NM_003740 (Hs), NM_021542 1497 NP_003731 (Hs) (SEQ ID NO: 228), channel TASK2 (Mm), NM_001039516 (Rn) NP_067517 (Mm), NP_001034605 (Rn) KCNK6 two pore domain potasium NM_004823 (Hs), 939 NP_004814 (Hs) (SEQ ID NO: 229), channel TWIK2 NM_001033525 (Mm), NP_001028697 (Mm), NP_446258 NM_053806 (Rn) (Rn) KCNK7 two pore domain potasium NM_033347 (Hs), NM_010609 921 NP_203133 (Hs) (SEQ ID NO: 230), channel member 7 (Mm) NP_034739 (Mm) KCNK9 two pore domain potasium NM_016601 (Hs), 1122 NP_057685 (Hs) (SEQ ID NO: 231), channel TASK3 NM_001033876 (Mm), NP_001029048 (Mm), NP_445857 NM_053405 (Rn) (Rn) KCNK10 two pore domain potasium NM_021161 (Hs), NM_029911 1614 NP_066984 (Hs) (SEQ ID NO: 232), channel TREK2 (Mm), NM_023096 (Rn) NP_084187 (Mm), NP_075584 (Rn) KCNK12 two pore domain potasium NM_022055 (Hs), NM_199251 1290 NP_071338 (Hs) (SEQ ID NO: 233), channel THIK2 (Mm), NM_022292 (Rn) NP_954859 (Mm), NP_071628 (Rn) KCNK13 two pore domain potasium NM_022054 (Hs), NM_146037 1224 NP_071337 (Hs) (SEQ ID NO: 234), channel THIK1 (Mm), NM_022293 (Rn) NP_666149 (Mm), NP_071629 (Rn) KCNK15 two pore domain potasium NM_022358 (Hs), 990 NP_071753 (Hs) (SEQ ID NO: 235), channel TASK5 NM_001030292 (Mm), NP_001025463 (Mm), NP_570826 NM_130813 (Rn) (Rn) KCNK16 two pore domain potasium NM_032115 (Hs), NM_029006 930 NP_115491 (Hs) (SEQ ID NO: 236), channel TALK1 (Mm) NP_083282 (Mm) KCNK17 two pore domain potasium NM_031460 (Hs) 996 NP_113648 (Hs) (SEQ ID NO: 237) channel TASK4-TALK2 KCNK18 two pore domain potasium NM_181840 (Hs), NM_207261 1152 NP_862823 (Hs) (SEQ ID NO: 238), channel TRESK1/TRESK2 (Mm), NM_001003820 (Rn) NP_997144 (Mm), NP_001003820 (Rn) Nucleotide-activated ion channels CNGA1 Cyclic nucleotide gated cation NM_000087 (Hs), NM_007723 2073 NP_000078 (Hs) (SEQ ID NO: 239), channel Alpha 1 (Mm), NM_053497 (Rn) NP_031749 (Mm), NP_445949 (Rn) CNGA2 Cyclic nucleotide gated cation NM_005140 (Hs), NM_007724 1995 NP_005131 (Hs) (SEQ ID NO: 240), channel Alpha 2 (Mm), NM_012928 (Rn) NP_031750 (Mm), NP_037060 (Rn) CNGA3 Cyclic nucleotide gated cation NM_001298 (Hs), NM_009918 2086 NP_001073347 (Hs) (SEQ ID NO: channel Alpha 3 (Mm), NM_053495 (Rn) 241), NP_034048 (Mm), NP_445947 (Rn) CNGA4 Cyclic nucleotide gated cation NM_001037329 (Hs), 1731 NP_001032406 (Hs) (SEQ ID NO: channel Alpha 4 NM_001033317 (Mm), 242), NP_001028489 (Mm), NM_053496 (Rn) NP_445948 (Rn) CNGB1 Cyclic nucleotide gated cation NM_001297 (Hs), NM_031809 3763 NP_001288 (Hs) (SEQ ID NO: 243), channel Beta1 (Rn) XP_001476248 (Mm), NP_113997 (Rn) CNGB3 Cyclic nucleotide gated cation NM_019098 (Hs), NM_013927 2434 NP_061971 (Hs) (SEQ ID NO: 244), channel Beta3 (Mm) NP_038955 (Mm) HCN1 hyperpolarization Cyclic NM_021072 (Hs), NM_010408 2674 NP_066550 (Hs) (SEQ ID NO: 245), nucleotide gated cation channel (Mm), NM_053375 (Rn) NP_034538 (Mm), NP_445827 (Rn) member 1 HCN2 hyperpolarization Cyclic NM_001194 (Hs), NM_008226 2671 NP_001185 (Hs) (SEQ ID NO: 246), nucleotide gated cation channel (Mm), NM_053684 (Rn) NP_032252 (Mm), NP_446136 (Rn) member 2 HCN3 hyperpolarization Cyclic NM_020897 (Hs), NM_008227 2326 NP_065948 (Hs) (SEQ ID NO: 247), nucleotide gated cation channel (Mm), NM_053685 (Rn) NP_032253 (Mm), NP_446137 (Rn) member 3 HCN4 hyperpolarization Cyclic NM_005477 (Hs), 3613 NP_005468 (Hs) (SEQ ID NO: 248), nucleotide gated cation channel NM_001081192 (Mm), NP_001074661 (Mm), NP_067690 member 4 NM_021658 (Rn) (Rn) TRP channels TRPC1 transient receptor potential NM_003304 (Hs), NM_011643 2284 NP_003295 (Hs) (SEQ ID NO: 249), (TRP) non selective cation (Mm), NM_053558 (Rn) NP_035773 (Mm), NP_446010 (Rn) channel C member 1 TRPC3 diacyglycerol (DAG) Ca2+ NM_003305 (Hs), NM_019510 2548 NP_003296 (Hs) (SEQ ID NO: 250), activated TRP channel C (Mm), NM_021771 (Rn) NP_062383 (Mm), NP_068539 (Rn) TRPC4 TRP receptor operated channel NM_016179 (Hs), NM_016984 2935 NP_057263 (Hs) (SEQ ID NO: 251), (Mm), NM_080396 (Rn), NP_058680 (Mm), NP_001076584 NM_001083115 (Rn) (Rn), NP_536321 (Rn) TRPC5 TRP receptor operated channel NM_012471 (Hs), NM_009428 2923 NP_036603 (Hs) (SEQ ID NO: 252), (Mm), NM_080898 (Rn) NP_033454 (Mm), NP_543174 (Rn) TRPC6 DAG activated TRP channel NM_004621 (Hs), NM_013838 2800 NP_004612 (Hs) (SEQ ID NO: 253), (Mm), NM_053559 (Rn) NP_038866 (Mm), NP_446011 (Rn) TRPC7 Cardiac TRP channel NM_020389 (Hs), NM_012035 2593 NP_065122 (Hs) (SEQ ID NO: 254), (Mm), XM_225159 (Rn) NP_036165 (Mm), XP_001067646 (Rn) TRPV1 Vanilloid (capsaicin) receptor NM_018727 (Hs), 2524 NP_061197 (Hs) (SEQ ID NO: 255), and noxious thermosensor NM_001001445 (Mm), NP_001001445 (Mm), NP_114188 channel NM_031982 (Rn) (Rn) TRPV2 noxious heat thermosensor NM_016113 (Hs), NM_011706 2299 NP_057197 (Hs) (SEQ ID NO: 256), channel (Mm), NM_017207 (Rn) NP_035836 (Mm), NP_58903 (Rn) TRPV3 warmth sensor channel NM_145068 (Hs), NM_145099 2380 NP_659505 (Hs) (SEQ ID NO: 257), (Mm), NM_001025757 (Rn) NP_659567 (Mm), NP_001020928 (Rn) TRPV4 Osmosensor channel NM_021625 (Hs), NM_022017 2620 NP_067638 (Hs) (SEQ ID NO: 258), (Mm), NM_023970 (Rn) NP_071300 (Mm), NP_76460 (Rn) TRPM1 putative melastatin TRP channel NM_002420 (Hs), NM_018752 4816 NP_002411 (Hs) (SEQ ID NO: 259), (Mm), NM_001037734 (Rn) NP_061222 (Mm), NP_001032823 (Rn) TRPM2 nucleotide sensing TRP channel NM_003307 (Hs), NM_138301 4513 NP_003298 (Hs) (SEQ ID NO: 260), (Mm), NM_001011559 (Rn) NP_612174 (Mm), NP_001011559 (Rn) TRPM3 Melastatin related TRP channel NM_020952 (Hs), 4669 NP_066003 (Hs) (SEQ ID NO: 261), NM_001035239 (Mm), NP_001030319 (Mm), XP_219902 XM_219902 (Rn) (Rn) TRPM4 Ca2+ activated Na+ TRP NM_017636 (Hs), NM_175130 3646 NP_060106 (Hs) (SEQ ID NO: 262), channel (Mm), NM_001136229 (Rn) NP_780339 (Mm), NP_001129701 (Rn) TRPM5 Nonselective, monovalent cation NM_014555 (Hs), NM_020277 3502 NP_055370 (Hs) (SEQ ID NO: 263), TRP channel (Mm), XM_344979 (Rn) NP_064673 (Mm), XP_344980 (Rn) TRPM6 Channel kinase TRP channel NM_017662 (Hs), NM_153417 6073 NP_060132 (Hs) (SEQ ID NO: 264), (Mm), XM_219747 (Rn) NP_700466 (Mm), XP_219747 (Rn) TRPM7 Kinase TRP channel NM_017672 (Hs), NM_021450 5602 NP_060142 (Hs) (SEQ ID NO: 265), (Mm), XM_001056331 (Rn) NP_067425 (Mm), XP_001056331 (Rn) TRPM8 Cooling and menthol-sensing NM_024080 (Hs), NM_134252 3319 NP_076985 (Hs) (SEQ ID NO: 266), TRP channel (Mm), NM_134371 (Rn) NP_599013 (Mm), NP_599198 (Rn) TRPA1 Nonselective cation TRP NM_007332 (Hs), NM_177781 3364 NP_015628 (Hs) (SEQ ID NO: 267), channel (Mm), NM_207608 (Rn) NP_808449 (Mm), NP_997491 (Rn) TRPP1 Nonselective cation TRP NM_000297 (Hs), NM_008861 2908 NP_000288 (Hs) (SEQ ID NO: 268), channel PKD2 (Mm), XM_573552 (Rn) NP_032887 (Mm), XP_573552 (Rn) TRPP2 Nonselective cation TRP NM_016112 (Hs), NM_181422 2422 NP_057196 (Hs) (SEQ ID NO: 269), channel PKD2L1 (Mm), NM_001106352 (Rn) NP_852087 (Mm), NP_001099822 (Rn) TRPP3 Nonselective cation TRP NM_014386 (Hs), NM_016927 1843 NP_055201 (Hs) (SEQ ID NO: 270), channel PKD2L2 (Mm), NM_001106156 (Rn) NP_058623 (Mm), NP_001099626 (Rn) TRPML1 Nonselective cation TRP NM_020533 (Hs), NM_053177 1747 NP_065394 (Hs) (SEQ ID NO: 271), channel (Mm), NM_001105903 (Rn) NP_444407 (Mm), NP_001099373 (Rn) TRPML2 Nonselective cation TRP NM_153259 (Hs), 1702 NP_694991 (Hs) (SEQ ID NO: 272), channel NM_001005846 (Mm), NP_001005846 (Mm), NP_080932 NM_026656 (Mm), (Mm), NP_001034094 (Rn) NM_001039005 (Rn) TRPML3 Nonselective cation TRP NM_018298 (Hs), NM_134160 1663 NP_060768 (Hs) (SEQ ID NO: 273), channel (Mm), NM_001012059 (Rn) NP_598921 (Mm), NP_001012059 (Rn) Chloride channels CLC1 chloride channel 1 NM_000083.2 2968 NP_000074.2 (SEQ ID NO: 274) CLC2 chloride channel 2 NM_004366.4 2698 NP_004357.3 (SEQ ID NO: 275) CLC3 chloride channel 3 NM_173872.3 2602 NP_776297.2 (SEQ ID NO: 276) CLC4 chloride channel 4 NP_001821.2 2284 NP_001821.2 (SEQ ID NO: 277) CLC5 chloride channel 5 NM_000084.2 2242 NP_000075.1 (SEQ ID NO: 278) CLC6 chloride channel 6 NM_001286.2 2611 NP_001277.1 (SEQ ID NO: 279) CLC7 chloride channel 7 NM_001287.4 2419 NP_001278.1 (SEQ ID NO: 280) Acid-sensitive cation channels ACCN1 amiloride-sensitive cation NM_183377.1 1696 NP_899233.1 (SEQ ID NO: 281) channel 1 ACCN2 amiloride-sensitive cation NM_020039.2 1726 NP_064423.2 (SEQ ID NO: 282) channel 2 ACCN3 amiloride-sensitive cation NM_020322.2 1600 NP_064718.1 (SEQ ID NO: 283) channel 3 ACCN4 amiloride-sensitive cation NM_018674.4 2005 NP_061144.3 (SEQ ID NO: 284) channel 4 Sodium channel auxiliary subunits SCN1B voltage gated sodium channel NM_001037.4 658 NP_001028.1 (SEQ ID NO: 285) type I b subunit SCN2B voltage gated sodium channel NM_004588.4 652 NP_004579.1 (SEQ ID NO: 286) type 2 b subunit SCN3B voltage gated sodium channel NM_018400.3 652 NP_060870.1 (SEQ ID NO: 287) type 3 b subunit SCN4B voltage gated sodium channel NM_174934.3 694 NP_777594.1 (SEQ ID NO: 288) type 4 b subunit Calcium channel auxiliary subunits CACNB1 voltage gated calcium channel NM_000723.4 1798 NP_000714.3 (SEQ ID NO: 289) b1 subunit CACNB2 voltage gated calcium channel NM_000724.3 1822 NP_000715.2 (SEQ ID NO: 290) b2 subunit CACNB3 voltage gated calcium channel NM_000725.3 1459 NP_000716.2 (SEQ ID NO: 291) b3 subunit CACNB4 voltage gated calcium channel NM_000726.3 1465 NP_000717.2 (SEQ ID NO: 292) b4 subunit CACNA2 voltage gated calcium channel NM_000722.2 (HS), 3277 NP_000713.2 (HS) (SEQ ID NO: 293), a2d subunit NM_001110843.1 (Mm) NP_001104313.1 (Mm), NP_037051 NM_012919 (RN) (RN) CACNG2 voltage gated calcium channel g NM_006078.3 (HS), 973 NP_006069.1 (HS) (SEQ ID NO: 294), subunit NM_007583.2 (Mm) NP_031609.1 (Mm) OTHERS ABCC8 ATP-binding cassette, sub- NM_000352.3 (HS) NP_000343.2 (HS) (SEQ ID NO: 295), family C SUR1 NM_011510.3 (Mm) NP_035640.2 (Mm) ABCC9 SUR2 NM_020297.2 NP_064693.2 (SEQ ID NO: 296) LGi1 LGi1 NM_005097.2 NP_005088.1 (SEQ ID NO: 297) CNTNAP2 CASPR2 NM_014141.5 NP_054860.1 (SEQ ID NO: 298) KCNAB1 Kv b1 NM_172160.2 NP_751892.1 (SEQ ID NO: 299) KCNAB2 Kv b2 NM_003636.3 NP_003627.1 (SEQ ID NO: 300) KCNAB3 Kv b3 NM_004732.2 NP_004723.2 (SEQ ID NO: 301) KCNMB1 BK b1 NM_004137.2 NP_004128.1 (SEQ ID NO: 302) KCNMB2 BK b2 NM_005832.3 NP_005823.1 (SEQ ID NO: 303) KCNMB3 BK b3 NM_171828.1 NP_741979.1 (SEQ ID NO: 304) KCNMB4 BK b4 NM_014505.5 NP_055320.4 (SEQ ID NO: 305) AQP1 AQP1 NM_198098.2 NP_932766.1 (SEQ ID NO: 306) AQP2 AQP2 NM_000486.5 NP_000477.1 (SEQ ID NO: 307) AQP3 AQP3 NM_004925.4 NP_004916.1 (SEQ ID NO: 308) AQP4 AQP4 NM_001650.4 NP_001641.1 (SEQ ID NO: 309) GJB1 Connexin 32 NM_000166.5, NM_000165.3 NP_000157.1 (SEQ ID NO: 310) GJA1 Connexin 43 NM_000165.3 NP_000156.1 (SEQ ID NO: 311) Hs: Homo sapiens (human) Mm: Mus musculus (mouse) Rn: Rattus norvegicus (rat)

In one particular embodiment of the invention, the membrane protein of interest is chosen from a GABA receptor, typically a GABA_(B) (GABA_(B)R1 or GABA_(B)R2, for example) receptor; a glutamate receptor, typically an alpha-amino-3-hydroxy-5-methylisoazole-4-propionic acid (AMPA) receptor, preferably the GluR3 subunit of the glutamate receptor (SEQ ID NO: 73 or SEQ ID NO: 74) or an N-methyl-D-aspartate (NMDA)-activated glutamate receptor, preferably the NMDAR1 subunit of the human glutamate receptor (SEQ ID NO: 62); aquaporin 2 (SEQ ID NO: 307); aquaporin 4 (SEQ ID NO: 309); a potassium channel; a calcium channel; an acetylcholine receptor, preferably the α3 subunit (SEQ ID NO: 9) of the human nicotinic acetylcholine receptor (ACHα3); a protein associated with voltage gated potassium channels, preferably CASPR2 (SEQ ID NO: 298) or LGI1 (SEQ ID NO: 297); a glycine receptor; connexin 32 (SEQ ID NO: 310); and connexin 43 (SEQ ID NO: 311).

Another subject of the invention relates to a chip comprising at least 20 different proteins of interest, for example at least 50, typically at least 100 different proteins of interest, preferably between approximately 20 and approximately 400 different proteins of interest, for example, approximately 250 or approximately 300 different proteins of interest, or between approximately 20 and approximately 100 different proteins of interest, for example, approximately 50 different proteins of interest, each protein of interest being optionally associated with one or more auxiliary subunits. These proteins of interest are preferably membrane proteins chosen from the proteins of which the sequences (preferably the sequences of the human proteins) are identified in Table 2.

The inventors have in particular developed chips comprising membrane proteins corresponding to proteins, such as ion channel proteins, transporter proteins and/or membrane receptor proteins which regulate the activity of an ion channel protein or of a transporter protein. These proteins are encoded by the sequences, preferably the sequences identified by a SEQ ID number, even more preferably by the human sequences identified by a SEQ ID number referenced in Table 2.

In order to fabricate these chips, the inventors have, without a priori, amplified by PCR a large number of subunits obtained from human brain cDNA (see Examples section). The products have been subcloned into an expression vector and transfected into cells in culture in multiwell plates, each well containing cells expressing a single type of membrane protein or membrane protein complex. The same tag sequence introduced into each channel makes it possible, in accordance with the knowledge of those skilled in the art, to measure and standardize the expression level of the proteins or protein complexes. The proteins and protein complexes of these cells are solubilized in a buffer containing a non-denaturing detergent. This solubilisate is then typically deposited in the form of a spot on a support, preferably a solid support, for example, on a solid membrane. Typically, various amounts of solubilisate, and also preferably one or more negative and positive controls, are deposited on the same support in order to verify the specificity of the signals obtained. The reading of the chip (detection of the antigen/antibody (Ag/Ab) complexes) can be carried out by immunofluorescence, immunoluminescence, or colorimetry by means of one of the techniques known to those skilled in the art.

Thus, a particular subject of the invention relates to a chip as described in the present text, said protein(s) of interest of which is (are) labelled with the same peptide sequence (tag), recognized by one or more detection antibodies known to those skilled in the art, such as the V5 antibody, which recognizes the GKPIPNPLLGLDST sequence (SEQ ID NO: 312). Other tags that can be used in the context of the present invention are, for example, the T7 Flag, Ha, myc and His (hexahistidine) tags.

In one preferred embodiment, the protein chip according to the invention comprises at least one positive control and/or one negative control, typically one positive control and one negative control, in order to verify the specificity of the signals obtained. The negative control typically consists of a cell extract not comprising the protein of interest. The positive control typically consists of human immunoglobulins, for example, IgGs, and makes it possible to validate the ability of the protein chip according to the invention to detect the presence of autoantibodies capable of binding to the proteins of interest in a biological sample to be tested.

The chips used in the context of the invention comprise a support capable of binding solubilized proteins, typically a solid (or semi-solid) support, preferably chosen from a membrane, typically a nitrocellulose or nylon membrane, glass, and a polymer capable of absorbing immune complexes, typically plastic (for example PVDF).

Such a support can be treated, in order to improve its cell extract-binding properties, using products and according to methods known to those skilled in the art. The support can, for example, be coated with a layer of organosilicon compounds (silane) and/or be functionalized with glycosaminoglycans (GAGs).

Another subject of the invention relates, moreover, to a kit comprising a chip as described previously, and, optionally, one or more reagents preferably chosen from a buffer for blocking the non-specific sites, a buffer allowing the association between antibodies and antigens (Ab/Ag), a washing buffer, one or more secondary antibodies, one or more product(s) for detecting said secondary antibody or antibodies, a standard solution for preparing a calibration curve, and instructions for use, typically instructions for use of one, several or all of said elements of the kit.

Such a kit may typically be used in the screening for new targets of interest, for example targets involved in the occurrence of an autoimmune disease, in particular of a disease affecting the nervous system of a mammal, or in the diagnosis or the monitoring of the progression of such an autoimmune disease in a patient receiving treatment in order to determine, in this patient, the efficacy of said treatment.

One particular subject of the invention thus relates to a kit for screening for targets of interest involved in the occurrence of a disease of interest, for example, of an autoimmune disease.

Another particular subject of the invention relates, moreover, to a kit for diagnosing a disease, typically an autoimmune disease as described previously, in an animal, typically a mammal, preferably a human being.

Preparation of the Chip

Another subject of the invention relates to a method for preparing a chip as described previously, comprising the following steps a) of cloning the cDNA(s) of interest; b) of expressing the protein(s), typically the membrane protein(s), encoded by said cDNA(s) in a protein-producing system, typically in cells in culture; c) of solubilizing the proteins expressed using a non-denaturing detergent, typically a non-ionic detergent (also identified as “mild detergent”), which enables the solubilization of said proteins while at the same time preserving their conformation (three-dimensional structure) and their ability to bind to a support; and d) of depositing said solubilized proteins on a support in order to obtain the chip of interest.

The invention also relates to a chip capable of being obtained by means of a method according to the invention as described previously.

Preferably, the chip according to the invention allows the detection, by those skilled in the art, of the proteins of interest which have bound to the support, by means of a conventional detection method, typically involving a single detection antibody. Such a chip allows its fabricator or its user to be sure that the protein(s) of interest is correctly expressed on said chip.

A means for obtaining such a chip consists of cloning the protein of interest (step a) of the method described previously) using an expression vector which allows i) the insertion of a tag, as described previously, in the same reading frame as the sequence encoding the protein of interest, and ii) the expression of a protein fused, preferably in the C-terminal position, to said tag. Such a tag makes it possible to verify and to measure the expression of the protein of interest associated with said tag.

The expression vector used is preferably a directional vector which allows the insertion of the gene of interest following PCR amplification thereof, without a prior enzymatic digestion step. An example of a directional vector is provided in the Examples section of the present application which uses the pcDNA6.2/V5/GW/D-TOPO vector (Invitrogen).

Advantageously, the vector used also benefits from the Gateway technology which makes it possible to envisage the rapid transfer of the gene encoding the protein of interest from a first vector to a second vector benefiting from the same technology but having different expression properties (the gene being cloned into the donor vector between two recombination sites compatible with all the Gateway recipient vectors). This technology makes it possible, for example, to transfer the gene encoding the protein of interest from a vector (donor vector) which allows expression in mammalian cells to a vector (recipient vector) which allows expression in insect cells, batrachian cells, yeast or bacteria, or else to a vector that is identical but has no tag. A vector benefiting from the Gateway technology can be chosen, for example, from the vectors pT-REx™-DEST30 (allowing expression in mammalian cells), pET™-DEST42 (allowing expression in bacteria), pDEST™8 (allowing expression in insect cells) and pYES2-DEST52 (allowing expression in yeasts).

During the PCR amplification of the genes, the forward primer preferably begins with the sequence CACC (SEQ ID NO: 313) (which precedes the start codon (ATG) of the gene of interest). In another preferred embodiment, a guanidine (G) nucleotide is inserted after the ATG start codon in order to obtain a Kozak sequence. In yet another embodiment, an additional GGA codon is introduced into the nucleotide sequence following the start codon of the gene of interest.

According to yet another preferred embodiment, the reverse primer does not contain a stop codon and/or does not end with the sequence complementary to the beginning of the forward primer.

Preferentially, the (forward and/or reverse) primers have a hybridization temperature of between approximately 70° C. and approximately 80° C., and also advantageously a percentage of GC units of less than approximately 60%.

Step b) of expressing the protein(s) encoded by said cDNA(s) of interest is advantageously carried out in cells in culture, preferably in human cells. The cells are generally placed in culture on multiwell plates, optionally treated to promote the adhesion of the cells using the techniques known to those skilled in the art, each well preferably containing cells expressing a single type of protein of interest. The expression of the protein of interest can be controlled as early as this stage, and, if required, standardized, through the possible detection (for example, by immunofluorescence) of the tag sequence that is expressed, where appropriate at the same time as the protein of interest. The cells selected are preferably cells exhibiting the satisfactory properties of adhesion to the culture support, which are simple to culture and to manipulate. Such cells can be advantageously selected, for example, from the HEK293A, COS, MDCK, CHO and CCL39 lines.

The method according to the invention comprises, moreover, a step c) of solubilizing the protein(s) of interest using a detergent, preferably using a non-denaturing detergent capable of preserving the conformation of the native protein and also its ability to bind to a support, typically to a support as described previously, preferably to a solid support of membrane type. The inventors have discovered that the detergent can be advantageously selected from Triton X-100, Triton X-114, Nonidet P-40, CHAPS, sodium cholate, sodium deoxycholate, digitonin, sarkosyl NL30 and octylglucoside.

Finally, the method comprises a step d) of depositing solubilized proteins of interest onto a support as described previously, in order to obtain the chip according to the invention. This step is typically carried out by depositing, in the form of spots, the solubilisates obtained at the end of step c) onto the chosen support, by means of techniques known to those skilled in the art.

Uses

There is, in particular, at the current time no tool for simply and effectively detecting the presence of autoantibodies directed against membrane proteins and/or membrane protein complexes in a biological sample, without having to visualize by microscopy the antigen/antibody (Ag/Ab) complexes formed at the surface of cells (previously transfected with the sequence encoding the antigen of interest) or of tissue sections. The chip according to the invention is thus the first available tool which allows the detection of Ag protein complexes or membrane protein/Ab complexes directly on a solid support (test on membrane, for example).

The chip according to the invention can be advantageously used in the context of a method for the diagnosis and/or for the monitoring of the progression of a disease, typically of an autoimmune disease as described previously, preferably of an encephalopathy associated with neuronal excitability disorders, linked to the expression of one or more of the proteins, in particular membrane proteins, of interest bound to the support of said chip, or else in the context of a method for determining the efficacy of a treatment applied to a given patient. The immediate or gradual disappearance of the autoantibodies sought in the biological sample originating from a patient receiving therapeutic treatment, evaluated using the protein chip according to the invention, thus makes it possible to verify, over time, the efficacy of the treatment applied to said patient. The absence of an effect of said treatment, readily detectable using said chip, makes it possible, moreover, to then correct or adapt, at an early stage, the treatment applied to a given patient.

The chip according to the invention may also be used in the context of methods required for searching for and identifying (screening methods) antibodies of interest, even present at very low concentrations (i.e., about one millimole), in particular autoantibodies, and also new antigen targets of diseases, for example, of autoimmune diseases (antigens of interest involved in the occurrence of an immune disease).

The biological samples from subjects identified as suffering from pathological conditions suspected of belonging or clearly identified as belonging to the autoimmune disease category can advantageously be used for this purpose.

It is thus now possible to produce and use a chip according to the invention (typically a chip which has i) all of the proteins, in particular of the proteins or membrane protein complexes, expressed by a given tissue, ii) all of the proteins, in particular of the proteins or membrane protein complexes, involved in a particular physiological mechanism, or iii) all of the proteins, in particular of the proteins or membrane protein complexes, involved in a specific metabolic pathway) in order to detect new autoantibodies or to identify new targets of therapeutic interest.

The large-scale screening permitted by this new tool allows the simultaneous detection of a very large number of Ag/Ab complexes. It also allows archiving of results which is simpler than that permitted by the currently known tests used on cells or tissue sections. The membranes represent surface areas of approximately 4 cm by 4 cm with the thickness of a sheet of paper. They are detected on a “photo” of identical size which can be stored like any paper or digital photo. In the case of tissue sections or transfected cells, the slides are archived in a freezer.

The facilitated identification of antibodies, for example of autoantibodies involved in autoimmune pathological conditions of the nervous system, and of their targets, using a chip according to the invention offering sufficient sensitivity, presently allows the development of diagnostic tests, in particular of serological tests, which are more efficient, simpler and less expensive.

The protein chip according to the invention, by virtue of the new antigen/antibody complexes that it makes it possible to identify, allows, moreover, the development of new therapeutic treatments involving the use of the antibodies identified, or of modulators of the formation of said antigen/antibody complexes, as medicaments, or in the context of the preparation of pharmaceutical compositions of interest.

The following figures and examples illustrate the invention by describing the obtaining of chips in accordance with the invention and by providing examples of uses of said chips, without limiting the scope thereof.

FIGURE LEGENDS

FIGS. 1A-1B: Validation of the efficiency of the protein chip by virtue of a test carried out on the samples originating from 43 patients.

A. Plan of a plate representing quadruplicates of a nitrocellulose membrane onto which are deposited, for each of the quadruplicates, decreasing concentrations of an extract of solubilized membranes of cells having overexpressed the NR1 receptor, an extract of solubilized membranes of cells without vector, and a 1/200^(th) dilution of human serum.

B. Photos of supports corresponding, respectively, to i) an expression control using the V5 tag alone, ii) an expression control using a sample originating from a positive patient, iii) an example of a negative sample, and iv) an example of a positive sample.

Of the 43 samples tested, 15 samples out of 17 positive samples were detected using the protein chip, i.e., a protein chip specificity of 88%. 100% of the negative samples were, moreover, detected.

FIGS. 2A-2F: Example of use of a protein chip according to the invention comprising 40 proteins of interest.

A. Plate 1: it contains two wells containing human IgGs and 22 wells containing the solubilized proteins of HEK cell membranes, each cell expressing a different membrane protein chosen from the proteins identified in Table 2 (referenced SEQ ID NO: 1 to SEQ ID NO: 311).

B. Plate 2: it contains two wells containing human IgGs and 22 wells containing the solubilized proteins of HEK cell membranes, each cell expressing a different membrane protein chosen from the proteins identified in Table 2 (referenced SEQ ID NO: 1 to SEQ ID NO: 311) and not expressed by the cells used to prepare plate 1.

C. Control plate (the correct expression is verified using the V5 tag alone).

D. Example of non-specific response.

E. Example of negative response.

F. Example of positive response.

EXAMPLES Example 1—Preparation of a Protein Chip According to the Invention and Developing of the Proteins Bound

Materials and Method

Cloning

The inventors used the pcDNA6.2-TOPO/GW/V5 expression vector (Invitrogen Cat#K2460-20). The majority of the primers were ordered from Eurogentec and the rest from Sigma.

The PCR is carried out on a Thermocycler machine (Biometra) with a commercial mixture of polymerases (HF enzyme mix from Fermentas cat#K0192). The amplification protocol is of the 2-step type with an extension and amplification temperature at 68° C.

The template for the amplification of the genes of interest is mainly derived from a cDNA library prepared from mRNA of healthy human brain (Invitrogen). The reverse transcription of this mRNA into cDNA is carried out using a SuperScript III (Invitrogen) according to the maker's protocol.

In the event of failed amplification of the gene of interest (either because of too little expression of this gene in the brain and, therefore, of the difficulty in amplifying it from this cDNA library, or because of the nucleotide nature of this gene, or else for another undetermined reason), another source of RNA (human cells of HEK type, for example) is used or a plasmid construct is used when it is available.

The PCR-amplified genes are separated from the dNTPs and from the template by (1%) agarose gel electrophoresis. The DNA bands of molecular weights corresponding to the genes of interest are cut out of the gel and purified on a column (of the “gel extraction purification” type from Qiagen, Promega or Macherey-Nagel). The amplified genes thus purified are then inserted into the expression vector according to the maker's guidelines (TOPO cloning, Invitrogen).

The plasmid constructs thus created are screened by bacterial transformation. Plated out on bacterial culture dishes, they are selected with an antibiotic of ampicillin type. The positive bacteria (of TOP10 type, Invitrogen) are placed in liquid culture the next day with the same antibiotic selection. This bacterial culture amplification makes it possible to amplify the amount of DNA available. This amplified DNA is purified on a column (of miniprep type, Qiagen). The plasmids that may have incorporated the gene of interest are sequenced using a sequencing platform. The sequencing primers are the T7 forward primer and a reverse primer specific for the pcDNA6.2-TOPO/GW/V5 vector. Only the beginning and the end of the sequence of the gene are verified by sequencing. This is because the subsequent detection of the presence of the tag (ta-V5g) in the cells in culture will by itself attest to the integrity of the protein.

Cell Culture

The inventors use the HEK293A human cell strain (Invitrogen Cat# R70507) as protein production system.

For the cell transfection, the inventors used jetPEI (Ozyme cat#101-10) as transfecting agent.

The inventors transfected a first series of cells on glass coverslips precoated with poly-L-Lysine in order to facilitate their adhesion.

Two days after the transfection, the inventors verify the presence of the Tag-V5 by immunofluorescence. The primary antibody directed against the V5 tag is a rabbit anti-V5 polyclonal antibody (Covance Cat#PRB189P). The anti-rabbit secondary antibody is coupled to a fluorophore of Alexa488 or Alexa594 type (Invitrogen). This detection finishes the validation of the cloning of the gene of interest.

A second cell culture series of dishes 60 mm in diameter, with the same transfecting agent (jet-PEI), makes it possible to produce a larger amount of cells. Two days after the transfection of the construct containing the gene of interest, the cells are recovered in the following way:

The culture medium is removed.

The cells are rinsed with 1 ml of PBS which is immediately suctioned off.

The cells are again rinsed with 1 ml of PBS.

They are left for 10 minutes at ambient temperature (AT).

All of the subsequent procedure is advantageously carried out in ice.

With a micropipette (P1000+filter tips), the cells are detached by repeated and careful projection of the PBS contained in the dish.

The resuspended cells are transferred into a 1.5 ml centrifugation tube (Eppendorf type).

The cells are centrifuged for 5 minutes at 14K RPM (revolutions per minute) and at 4° C. in a bench centrifuge.

The supernatant is removed.

The cells are resuspended in 50 μl of solubilization buffer prepared, for example, as follows:

40% glycerol, 2% CHAPS, 140 mM NaCl, 2 mM EDTA, 20 mM Tris, pH8.8, in PBS to which an anti-protease mix of “Complete” type (from Roche) has been added.

The cells are solubilized in this buffer by pipetting up and down approximately 50 times. Still in the microfuge tube, the solubilisate is left in ice for one hour.

A centrifugation is carried out for 10 minutes at 2500 RPM (revolutions per minute), and at 4° C.

The supernatant is transferred into a clean tube, DNA loading buffer is added, and the mixture is stored at −80° C.

Once the collection of several solubilisates of various cultures and of various expressed proteins has been established, these soluble proteins are distributed into conical-bottom 96-well plates, advantageously using the 24 central wells.

While maintaining these plates on ice, a fraction of each solubilisate is sampled using a multiwell replicator and a footprint is produced in triplicate on a nitrocellulose membrane (Hybond-C Extra Cat#RPM303C, Amersham).

This membrane constitutes the support for the 24 solubilisates to be tested with the sample from a patient. An identical footprint is produced in order to verify the correct expression of the V5-tagged proteins in each solubilisate.

On the day of the test, the footprinted membranes are cut to the size limit of the 24 wells. (One corner is notched in order to orient the membrane).

This membrane is rolled up and introduced into a 5 ml hemolysis tube.

It is ensured that the entire surface area of the membrane is uniformly distributed inside the tube.

This membrane is incubated for 15 minutes with 2 ml of blocking solution (PBS, 5% milk, 0.2% NP40).

This blocking medium is replaced with 750 μl to 1 ml of antibody solution at 1/20^(th) (50 μl of CSF in 1 ml of blocking solution). Everything is incubated overnight at ambient temperature on a rotary machine.

The incubation medium is recovered and frozen for a second use.

Each membrane is rinsed three times in the hemolysis tubes with the rinsing solution (PBS, 0.1% NP40).

Incubation is carried out for 2 hours at ambient temperature with the secondary antibody (diluted to 1/5000^(th) in the blocking solution (PBS, 5% milk, 0.2% NP40)). The secondary antibody is coupled to HRP (horseradish peroxidase) (anti-human and anti-rabbit from Jackson ImmunoResearch).

Rinsing is carried out three times with the rinsing solution (PBS, 0.1% NP40) and once with PBS alone.

The blots are developed with 500 μl of ECL (enhanced chemiluminescence) (Pierce) in the developing machine (FujiFilm Fluorescent Image Analyzer FLA-3000).

Example 2—Validation of the Efficiency of the Protein Chip

In the present example, samples (of CSF type) originating from a cohort of patients known to have antibodies directed against the NMDA receptor subunit NR1 (initially detected using an immunocytology method) and also samples originating from individuals not suffering from an autoimmune disease are used to validate the efficiency of the protein chip according to the invention.

The invention was validated by means of a blind test carried out on various samples originating from 17 patients known to contain anti-NR1 antibodies (in a population consisting of 26 healthy individuals).

According to the protocol described above, the membrane proteins of cells expressing the NR1 receptor are solubilized and deposited on a nitrocellulose membrane. Each membrane has four deposits of the protein of interest corresponding to four decreasing dilutions of the same solubilized membrane protein preparation resulting from cells overexpressing the NR1 subunit, one control deposit corresponding to solubilized membrane proteins from cells expressing the empty vector, and one control deposit corresponding to a solution of human serum from a healthy subject (see FIG. 1). After incubation and detection as described above, the results show that, of the 17 afflicted patients, the test recognized 15 thereof and therefore has an efficiency of 88%. The specificity for the negative patients is 100%. The two false positives recognized by the protein chip according to the invention correspond to two candidates who had been detected as positive and then discarded in a second screening of a first test carried out on cells.

Example 3—Screening Using the Protein Chip

In the present example, the invention was tested on a cohort of 50 patients suffering from neurological disorders of unknown origin (and which were potentially autoimmune) for whom the inventors sought to know whether they had antibodies directed against one or more proteins among 40 proteins deposited on a nitrocellulose membrane.

In this example, each plate (FIG. 2) possesses 20 samples of solubilized membrane proteins from cells individually overexpressing 20 proteins of interest (codified here in A15, F28, F31, etc.) and also two samples corresponding to a dilution of human serum from a healthy subject.

The deposits on nitrocellulose membrane were carried out according to the protocol described above (with a replicator) in duplicate and while combining the deposits of the two plates (plate 1 at the bottom, plate 2 at the top) on the same membrane (see FIG. 2).

The results show that, among the 50 patients tested, many patients were negative and therefore did not have antibodies directed against one of the 40 proteins deposited, a small number had antibodies against all the proteins deposited, and one patient had antibodies directed specifically against one protein (H7).

After comparison of these results with the clinical picture of the cohort of 50 patients, it appears that the non-specific response of the patients having antibodies directed against all the proteins is partly explained by the degree of advancement and of severity of their disease, since most of them died even before the test was set up (massive and non-specific immune response). On the other hand, the sole result regarding the H7 protein is the perfect example of the possible identification, using the chip according to the invention, of the association (demonstrated for the first time) that exists between a particular channel (protein identified in the present example as the H7 protein) and a neurological disease. 

The invention claimed is:
 1. A method for preparing a protein chip comprising the following steps: cloning one or more cDNA(s) of interest encoding one or more membrane proteins expressed in the cells of the nervous system of an animal, and which is (are) involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders, using an expression vector which allows i) the insertion of a tag, in the same reading frame as the sequence encoding the one or more membrane proteins, and ii) the expression of the one or more membrane proteins fused to said tag, expressing the one or more membrane proteins encoded by said cDNA(s), fused to said tag, in cells in culture, solubilizing the expressed one or more membrane proteins using a non-denaturing detergent which allows the solubilization of said one or more membrane proteins while at the same time preserving their conformation and their ability to bind to a support, and depositing said solubilized one or more membrane proteins on a support, thereby obtaining the chip of interest.
 2. The method according to claim 1, wherein said one or more solubilized membrane proteins are selected from the group consisting of an ion channel protein, a transporter protein, and a membrane receptor protein which regulates the activity of an ion protein channel or of a transporter protein.
 3. The method according to claim 1, wherein said at least one or more membrane proteins comprise a protein selected from SEQ ID NO: 1 to SEQ ID NO:
 311. 4. The method according to claim 1, wherein the one or more membrane proteins comprise a protein selected from the group consisting of a GABA receptor; a glutamate receptor; aquaporin 2 (SEQ ID NO: 307); aquaporin 4 (SEQ ID NO: 309); a potassium channel; a calcium channel; an acetylcholine receptor, a protein associated with voltage gated potassium channels; a glycine receptor; connexin 32 (SEQ ID NO: 310); and connexin 43 (SEQ ID NO: 311).
 5. The method of claim 4, wherein the one or more membrane proteins comprise an N-methyl-D-aspartate (NMDA)-activated glutamate receptor.
 6. The method of claim 5, wherein the NMDA-activated glutamate receptor is the NMDAR1 receptor (SEQ ID NO: 62).
 7. The method according to claim 1, wherein the support upon which the membrane proteins are deposited comprises: a) at least 100 different membrane proteins of interest; b) between approximately 200 and approximately 400 different proteins of interest; c) approximately 250 different proteins of interest; or d) approximately 300 different proteins of interest, wherein each protein of interest is optionally associated with one or more auxiliary subunits.
 8. The method according to claim 1, wherein said detergent is selected from the group consisting of Triton X-100, Triton X-114, Nonidet P-40, CHAPS, sodium cholate, sodium deoxycholate, digitonin, sarkosyl NL30 and octylglucoside.
 9. The method according to claim 1, wherein said method does not require an ultracentrifugation step.
 10. The method according to claim 1, wherein said method does not comprise a fractionation step using salts. 